Fermt1, Smg1), recovery from DNA damage  and regulation of heterochromatin assembly . Non-irradiated, senescent oocytes exhibit not only high chromocenter mobility and satellite distension but also a high frequency of extra chromosomal satellite DNA. Notably, analysis of biological aging using an oocyte-specific RNA clock revealed cellular communication, posttranslational protein modifications, chromatin and histone dynamics as the top cellular processes that are dysregulated in both senescent and irradiated oocytes. Our results indicate that unfolding of heterochromatin fibers following acute genotoxic stress or cellular aging induced the formation of distended satellites and that abnormal chromatin structure together with increased chromocenter mobility leads to chromosome instability in senescent oocytes.The mechanisms leading to changes in mesoscale chromatin organization during cellular aging are unknown. Here, we used transcriptional activator-like effectors, RNA-seq and superresolution analysis to determine the effects of genotoxic stress on oocyte chromatin structure. Major satellites are organized into tightly packed globular structures that coalesce into chromocenters and dynamically associate with the nucleolus. Acute irradiation significantly enhanced chromocenter mobility in transcriptionally inactive oocytes. In transcriptionally active oocytes, irradiation induced a striking unfolding of satellite chromatin fibers and enhanced the expression of transcripts required for protection from oxidative stress ( Super-resolution imaging and RNA clock analyses show that exposure of mouse oocytes to ionizing radiation compromises chromatin organization and dramatically accelerates biological aging. Heterochromatin domains exhibit profound conformational changes during cellular senescence with dire consequences for genome integrity, but little is known about the underlying mechanisms3. Senescent cells undergo dramatic changes in genome organization, potentially altering chromatin conformation and chromosome packaging at different scales5. For example, chromatin conformation (Hi-C) assays revealed that both senescent human fibroblasts and Hutchinson-Gilford progeria cells exhibit a relaxation of chromatin topographical organization with a reduction of local interactions between topologically associated domains (TADs) in the human genome5,7.In mammalian cells, pericentric heterochromatin is essential for higher-order chromosome organization, nuclear architecture, and maintenance of genome integrity4. Elegant studies suggest that SADs formation represents a large-scale unfolding of chromatin, above the level of the nucleosome4. However, the mechanisms involved in regulating such striking changes in mesoscale chromatin structure are not clear at present. In contrast with our current understanding of nucleosome organization in the human and mouse genomes8, little is known about the mechanisms that regulate mesoscale chromatin organization from the canonical 10 nm fiber into a highly compact metaphase chromosome. Importantly, the regulation of some of the most critical levels of large-scale chromatin organization, as well as the response of centromeric heterochromatin domains to developmental or environmental transitions remains poorly understood.Senescence-associated distension of satellite DNA (SADs) is an early senescence marker consistently detected in human and mouse models of cellular aging, suggesting that heterochromatin reorganization may be an integral component of the senescence process10. In fully-grown oocytes at the germinal vesicle (GV) stage, chromatin is initially found in a highly decondensed state to sustain high levels of nascent transcription, a configuration called non-surrounded nucleolus (NSN)12. However, in preparation for meiotic resumption, oocytes undergo a striking change in nuclear organization with formation of a perinucleolar chromatin rim or karyosphere, a configuration known as surrounded nucleolus (SN)11. Chromatin remodeling into the SN configuration is also associated with a concomitant global repression of nascent transcription12. Importantly, these critical developmental transitions in chromatin organization are highly conserved between human and mouse ova13. The large size of the mammalian oocyte nucleus and the potential to analyze chromatin in the presence or absence of transcription provides a natural in vivo model to dissect the molecular pathways of chromatin remodeling at critical developmental milestones.Direct visualization of 3D-genome organization in space and time and with a level of resolution beyond the optical diffraction limit is essential to understand the biological principles of chromatin organization, chromatin fiber folding, and chromosome compaction in the mammalian genome. Functional differentiation of chromatin structure in pre-ovulatory oocytes provides a prominent example of large-scale chromatin remodeling with direct implications for chromosome stability and developmental potential14 for live-cell chromocenter tracking, superresolution structured illumination15, and RNA sequencing to determine the effects of genotoxic stress induced by acute γ-irradiation or reproductive senescence on large-scale chromatin organization. Our results indicate that in transcriptionally inactive (SN) oocytes, chromocenters are extremely mobile and dynamically interact with the nucleolus. Both acute irradiation and cellular aging significantly increased chromocenter mobility in SN oocytes but resulted in a striking distention of satellite DNA in transcriptionally active (NSN) oocytes. Notably, senescent oocytes exhibit not only severe distension of satellite DNA but also a high frequency of extra chromosomal DNA fragments. Our results indicate that both increased chromocenter mobility and abnormal chromatin structure contribute to the mechanisms of chromosome instability in senescent oocytes.Here, we use transcriptional activator-like effectors (TALEs) against major satellite sequences17. In the mouse genome, pericentric heterochromatin (PCH) domains are formed by 6 megabases of repetitive major satellite DNA adjacent to the centromere of each chromosome18. However, little is known about the role of PCH in the regulation of large-scale chromatin organization and nuclear architecture. Microinjection of a TALE-effector14 into live, GV stage oocytes revealed that major satellite sequences (green) aggregate into large globular domains or chromocenters in oocytes that exhibit the NSN configuration  indicating that at this stage, most chromocenters are formed by interactions between two homologous chromosome bivalents but also occasionally by a single homologous chromosome bivalent  and minor (red) satellite sequences, which label PCH domains and centromeres, respectively Fig. . Detectient Fig. . These r15. SR-SIM resolves the mesoscale organization of major satellite DNA beyond the optical diffraction limit and reveals a network of tightly packed chromatin fibers (green) that also exhibit bright H2B signal (red)  Fig. ; Inset. ed) Fig. . Notablyed) Fig. ; arrow. 3) and SN (6.04 ± 5.6 μm3) oocytes revealed a significant reduction (P < 0.001) in chromocenter volume in SN oocytes  increased significantly (p < 0.0001) to 0.005 μm/s in 71 chromocenters tracked in irradiated oocytes (n = 10), resulting in a mean total path length of 56.5 microns in controls and 89.9 microns in irradiated oocytes  chromocenters in non-irradiated senescent oocytes (n = 11) revealed high chromocenter mobility resulting in a total path length of 76–115 microns. The average chromocenter speed (0.005 μm/s) in aged oocytes showed no significant differences with that observed in irradiated, young oocytes  SN oocytes to a 5 Gy dose of acute γ-irradiation and compared chromatin dynamics with non-irradiated, senescent (>10-month-old) oocytes. Live-cell imaging revealed that chromocenters in control young, non-irradiated SN oocytes exhibit a path length of 55–71 microns in a 5 h period Fig. . In conttes Fig. . Notablytes Fig. , resulti19. SR-SIM analysis revealed that γ-irradiation results in a striking chromocenter decondensation after 24 h in oocytes that exhibit the NSN configuration, Notably, ATRX remained bound to decondensed chromocenter chromatin fibers in irradiated oocytes  of the nucleus of NSN oocytes prior to and 24 h after irradiation. In control non-irradiated oocytes, euchromatin is organized into a lose network of DNA fibers that is widely distributed and occupies the entire volume of the germinal vesicle Fig. . Maximumers Fig. .Fig. 4Di3  revealed that the volume of chromocenters in control NSN oocytes ranges between 3 to 35 μm3 Fig. . However. 3 Fig. . Moreove. 3 Fig. . These r10. Analysis of differentially expressed transcripts revealed significantly (P < 0.05) enhanced expression of 120 transcripts and a significant downregulation of 115 transcripts 24 h after irradiation compared with control non-irradiated oocytes. Notably, the zinc finger protein (Zfp296) exhibited the highest statistical significance and showed a 6.1-fold overexpression in irradiated oocytes. Zfp296 is a component of mouse PCH that interacts with heterochromatin proteins, including ATRX and contributes to the regulation of histone modifications at major satellite repeats21. In addition, the actin-related protein 3 (Actr3) which functions in F-actin polymerization, nuclear lamina assembly and chromatin reorganization22 exhibited a significant (p < 0.05) 11.8-fold overexpression. Kbtbd8, a ubiquitin ligase involved in free-radical control and oocyte quality23, the Ski proto-oncogene, which directly binds to and is required to regulate histone modifications at major satellites24 and the solute carrier (Slc7a2), that potentially interacts with the Ski proto-oncogene24 were all upregulated >20 fold in irradiated oocytes (p < 0.05). Moreover, transcripts required for protection from oxidative stress 26 and recovery from DNA damage 28 showed a significant (P < 0.05) overexpression. In contrast, amongst the most significantly downregulated transcripts, cingulin (Cgn) a tight junction protein known to cause activation of cellular senescence pathways following its functional ablation29, Birc6/Bruce, an inhibitor of apoptosis30 that regulates DNA double-strand break response31 and is required for genome stability32 as well as the N-Ras proto-oncogene, which is required to modulate the DNA damage response33 were all downregulated >20 fold. In addition, Rev1 a translesion synthesis protein that regulates radiation resistance34 and Dnmt3l were downregulated >5 fold. Gene ontology (GO) analysis of upregulated transcripts indicates roles in cellular processes such as RNA import into nucleus, regulation of mitotic sister chromatid separation, positive regulation of heterochromatin assembly and nuclear matrix organization while GO terms for cellular function revealed a role in histone deacetylase inhibitor activity, box C/D snoRNP complex binding, box H/ACA snoRNP complex binding and nitric oxide synthase activity  of 78.8 days in cross validation  oocytes exhibit unfolding of chromatin fibers, and a striking distension of satellite DNA following irradiation. This process is associated with enhanced expression of factors required for protection from oxidative stress, recovery from DNA damage and regulation of heterochromatin assembly. Notably, non-irradiated senescent oocytes exhibit inherently high chromocenter mobility and distension of satellite DNA. Together, our results indicate that increased chromocenter mobility and abnormal chromatin fiber folding at PCH result in the formation of extrachromosomal DNA fragments in senescent oocytes and contribute to the mechanisms of chromosome instability during oocyte aging.Pericentric heterochromatin is essential for chromosome stability during female meiosis37. The number and distribution of chromocenters are cell-type specific and reflect the epigenetic state acquired during differentiation38. In somatic cells, major satellites from several chromosomes associate to form a chromocenter. Notably, the centromeres of each chromosome remain as two separate foci that can be clearly resolved by SR-SIM39. In contrast, superresolution analysis of both minor and major satellites in oocytes revealed the presence of fused centromeres on each homologous chromosome bivalent and suggests that large chromocenters are formed by the coalescence of major satellites from several homologous chromosome bivalents. Thus, by bringing multiple homologous chromosomes together at each chromocenter, PCH may directly affect the formation and stability of long-range chromosome interactions in the oocyte genome. The mechanisms regulating the association of major satellite DNA between different chromosomes are not known. Major satellite clustering may be mediated by heterochromatin binding proteins, epigenetic modifications, and potentially non-coding RNAs41. However, the specific factors that mediate the coalescence of satellite DNA into chromocenters remain to be determined. Notably, an increase in chromocenter number and a concomitant reduction in their volume in SN oocytes suggest the presence of a developmental transition whereby chromocenters become reorganized in preparation for meiotic cell division to ensure accurate chromosome segregation.Repetitive DNA constitutes >40% of the mammalian genome and is critical for chromatin organization42. Consistent with that study, SR-SIM resolves highly compact heterochromatin fibers in the oocyte chromocenters. Importantly, in contrast with electron spectroscopic imaging tomography, SR-SIM is compatible with protein localization and resolved the association of ATRX with individual major satellite DNA fibers. Loss of ATRX function is associated with centromere instability during oocyte meiosis43 consistent with a role in the regulation of heterochromatin structure. However, the specific factors regulating chromatin fiber folding and fiber–fiber density in the oocyte chromocenters remain to be determined.Partial 3-D reconstruction using electron spectroscopic imaging in mouse fibroblasts revealed that chromocenter formation involves an increase in chromatin fiber density and compact chromatin fiber folding40. Our results indicate that oocyte chromocenters exhibit high mobility and establish dynamic interactions with the nucleolar surface. In SN oocytes, some chromocenters exhibited a combination of linear and non-linear trajectories with a total path length of up to 60–70 μm, suggesting remarkable chromocenter dynamics compared with somatic cells. For example, stochastic analysis of relative trajectories indicates that in human cells, centromeres exhibit constrained relative motion within a few microns44. In HeLa cells, centromeres have been shown to remain mostly stationary but can occasionally move at speeds up to 7–10 μm/hour45. Importantly, it has been suggested that short range, constrained centromere motion is critical to prevent the loss of chromosome folding44. Our results provide evidence indicating that oocyte chromocenters fuse and split and that dynamic interactions between PCH domains of different chromosomes also take place at the nucleolar surface. Thus, regulation of heterochromatin dynamics is of direct consequence for genome integrity46.In somatic cells, the spatial organization of chromocenters is regulated by interactions with the nucleolus52. Our results indicate that DNA damage can also induce large-scale movements of heterochromatin domains in the oocyte genome.Acute γ-irradiation significantly increased chromocenter mobility in SN oocytes due to detachment from the nucleolus and changes in large-scale chromatin organization. Within 30 min, the patterns of chromocenter oscillatory movements changed into a linear displacement, resulting in a significant increase in the average chromocenter speed and total path length suggesting that genotoxic stress enhanced global chromatin mobility. In somatic cells, changes in chromatin motion are an integral component of the cellular response to DNA damage and specific genomic loci exhibit increased mobility over long distances in order to facilitate the repair of double-strand breaks53. However, the mechanisms of SADs formation are not fully understood. Our results indicate that γ-irradiation induced distension of major satellites within 24 h in transcriptionally active (NSN) oocytes due to large-scale unfolding of chromatin fibers. ATRX is known to interact with HP1 in heterochromatin formation and is critical for centromere stability in mouse oocytes55. However, ATRX remained associated with PCH fibers in irradiated oocytes suggesting that unfolding of major satellite DNA takes place in spite of ATRX localization. Consistent with this observation, a recent study indicates that neither the size, nor the state of chromatin compaction at chromocenters is dependent on the levels of HP1 in mouse fibroblasts41. Notably, Fortuny et al. recently demonstrated a significant reduction in PCH compaction following targeted UV damage56. However, several repressive heterochromatin marks persist at decondensed chromocenters suggesting that retention of remodeling factors may be part of a maintenance mechanism to safeguard heterochromatin organization and facilitate recovery from DNA damage56.In human and mouse somatic cells, SADs formation is a marker of early senescence associated with changes in large-scale chromatin organization53. In mouse fibroblasts, chromocenters resemble collapsed polymer globules that can adopt condensed or decondensed folding states reflecting important mechanisms of chromatin organization41. The factors responsible for regulating chromatin fiber folding and fiber–fiber cross-links to maintain high-density DNA packing remain to be established. However, transcriptome analysis of irradiated oocytes indicates that unfolding of major satellite DNA fibers is associated with changes in factors required for regulation of heterochromatin formation. Transcripts for the zinc finger protein Zfp296 exhibited a 6-fold overexpression. Zfp296 is localized to pericentric heterochromatin in mouse post-implantation embryos where it acts as a negative regulator of H3K9me321. However, γ-irradiation also induced a 20-fold overexpression of transcripts for the Ski proto-oncogene. Ski localizes to PCH and is required to maintain the levels of H3K9me3 and transcriptional repression of several genes  located within major satellites of chromosome 924. Interestingly, Ski overexpression induced desensitization of human epithelial cells to genotoxic drugs57 suggesting a potential role in protection from DNA damage. Our results indicate that γ-irradiation may disrupt the balance between expression of positive and negative regulators of histone modifications and transcriptional silencing of PCH domains thus interfering with the establishment of critical epigenetic marks required for chromocenter compaction in the oocyte genome.Chromocenters are formed by groups of chromatin fibers that organize into a densely packed and highly compact structure22. Transcripts for Actr3 were overexpressed >10 fold following oocyte irradiation, potentially contributing to changes in large-scale chromatin organization and suggesting that changes in chromocenter dynamics may be mediated by abnormal interactions with the nuclear lamina. Importantly, our results revealed that coincident with changes in mesoscale chromatin organization after irradiation, the oocyte also triggers an active transcriptional response to DNA damage. Consistent with this notion, we detected a significant overexpression of factors required for protection from oxidative stress , restoration of chromatin structure at DNA breaks (Tlk2), DNA repair and maintenance of genome stability (Rad54l). The human FERMT-1 gene encodes for Kindlin-125. Kindlin-1 plays a key role in regulating the cellular response to acute oxidative stress25. Notably, it is required for mitotic spindle assembly58. Smg1 functions in the response to genotoxic stress, DNA damage, and maintenance of telomere integrity26. The Tousled-like kinase 2 (Tlk2) is critical for recovery following DNA damage at the G2 phase where it regulates the histone chaperone Asf1A to restore histone H3 deposition into damaged chromatin27. The DNA translocase RAD54l promotes accurate repair of DNA damage to prevent chromosome segregation defects28. Notably, γ-irradiation also induced transcriptional changes associated with the activation of cellular senescence and regulation of chromatin structure . Amongst the most significantly downregulated factors, we detected a >20-fold reduction in transcripts for Cingulin, a tight junction protein localized to the sites of contact between cumulus cells and the oocyte cytocortex59. In Zebra fish embryos, loss of Cingulin activates cellular senescence pathways29. Transcripts for Rev1 were downregulated >5 fold. Notably, inhibition of Rev1 is associated with activation of senescence in human ovarian cancer cell lines and mouse fibroblasts60. Transcripts for Birc6/Bruce were downregulated >20 fold. Birc6 is required for homologous recombination in somatic cells and prevents non-homologous chromosome interactions during spermatogenesis32. Collectively, our results indicate that γ-irradiation activates signaling pathways associated with the onset of cellular senescence as well as repair from DNA damage and suggest a mechanistic link between the DNA damage response and regulation of large-scale heterochromatin structure in the oocyte genome. Studies are in progress to identify the specific protein factor(s) or potential histone post-translational modifications that induce the loss of chromocenter compaction in NSN oocytes.The actin-related kinase (Actr3) maintains chromatin organization and influences chromatin dynamics through its role in the regulation of nuclear lamina integrity4. More recently, STED microscopy revealed that SADs are formed by globular domains linked by threads of distended DNA53. Our direct visualization of major satellite chromatin fibers in mouse oocytes revealed that changes in chromocenter compaction are due to both, unfolding of chromatin fibers and increased fiber–fiber distance that result in a significant increase in chromocenter volume.We provide evidence indicating that non-irradiated senescent oocytes with the SN configuration exhibit similar chromocenter mobility compared to irradiated oocytes. Notably, senescent oocytes that remained at the NSN configuration also exhibited distension of major satellite sequences. This suggests that acute genotoxic stress following γ-irradiation and chronic genotoxic stress resulting from cellular aging induce major satellite distension. Comparison of the cellular mechanisms that are dysregulated in both senescent and irradiated oocytes using an RNA clock revealed that abnormal cellular communication, chromatin dynamics, and histone posttranslational modifications are important mechanisms associated with the formation of abnormal chromatin structure in both senescent and irradiated oocytes and resulted in the advanced biological age detected in irradiated oocytes. Previous studies suggested that SADs formation in human senescent cells may be due to abnormal chromatin folding61. Our SR-SIM analysis provides evidence that NSN senescent mouse oocytes exhibit distension of major satellites, abnormal chromatin fiber folding and disrupted large-scale chromatin organization. To the best of our knowledge, this is the first report of abnormal chromocenter formation during in vivo oocyte senescence. Our results are consistent with the notion that major satellite distension is due to changes in higher-order packaging of heterochromatin. The factor(s) responsible for maintaining the highly compact state of chromocenters and packing density of satellite DNA fibers remain to be established. However, the differential response to both γ-irradiation and senescence observed between NSN and SN oocytes provides intriguing new insight. Indeed, in irradiated SN oocytes chromocenters remain highly compact, albeit with increased mobility suggesting that the factor(s) responsible for regulating major satellite fiber packing and chromocenter condensation are probably lost from irradiated or senescent NSN oocytes but remain bound to chromocenters in SN oocytes. Thus, it is conceivable that this may reflect a previously unappreciated step in the epigenetic maturation of heterochromatin domains in pre-ovulatory oocytes. The effect(s) of SADs formation on centromere structural integrity and chromosome segregation are not fully understood. However, our recent studies in senescent mouse fibroblasts indicate that SADs formation is associated with centromeric breaks and severe chromosome instability3. Consistent with this notion, a high proportion of senescent oocytes exhibit extrachromosomal satellite DNA fragments, a sign of chromosome instability. Notably, the co-localization of H2B and extrachromosomal major satellite DNA indicates that these fragments are still packed within nucleosome-containing chromatin. Previous studies indicate that precocious bivalent separation may be the primary defect in age-related aneuploidy62. However, the mechanisms involved in this process remained unclear. Our studies provide evidence indicating that abnormal pericentric heterochromatin structure, increased chromocenter mobility and distension of satellite DNA contribute to the formation of extrachromosomal satellite DNA and chromosome instability, predisposing to abnormal chromosome segregation in senescent oocytes. Our findings have implications not only for the field of onco-fertility but also to determine the transcriptional and epigenetic mechanisms of oocyte senescence and genome instability.A previous study using diffraction limited imaging detected an indiscernible chromatin configuration in aged mouse oocytesAnimal use protocols were approved by the ‘Institutional Animal Care and Use Committee’ (IACUC) of the University of Georgia, and all experiments were conducted in accordance with guidelines. Mice were housed at a constant temperature (24–26 °C) and a controlled light cycle (12 h light/dark), with food and water provided ad libitum.2, 5%O2, and balanced N2.All experiments involved GV stage oocytes collected from non-primed female mice with a C57/Bl6/DBA2J F1 hybrid background between postnatal days 16 to 24 as well as from aged females (10 months). The oocytes were recovered from the ovaries by follicle puncture and cumulus oocyte complexes were transferred to minimal essential medium (MEM) supplemented with 3 mg/ml bovine serum albumin  and milrinone  to prevent meiotic resumption. Surrounding cumulus cells were removed by gentle pipetting followed by several rinses in fresh medium. Oocytes were cultured for specified periods of time and maintained at 37 °C in MEM/BSA plus milrinone under 5% CODenuded oocytes were fixed at indicated timepoints post irradiation and permeabilized in 2% paraformaldehyde in PBS supplemented with 0.1% Triton X-100 for 20 min at room temperature before washing and blocking in PBS supplemented with 5% serum and 0.05% Triton X-100 overnight at 4 °C. Oocytes were immunolabeled with specific antibodies against the DNA double-strand break marker γH2AX , and the heterochromatin marker ATRX . In brief, fixed oocytes were incubated with the primary antibodies diluted in blocking buffer overnight at 4 °C, then washed and incubated with specific Alexa Fluor 488 or 555 conjugated secondary antibodies  for 1 h at 37 °C. After a series of final wash steps in blocking buffer, the oocytes were transferred onto glass slides and overlaid with mounting medium  containing DAPI  to counterstain the DNA.63 and pTALYM3B15 (Addgene cat# 47878)19. In brief, 10 pl capped mRNA were microinjected into the cytoplasm of GV-intact oocytes in MEM medium containing 1 μg/ml milrinone using an electronic FemtoJet microinjector (Eppendorf) and Eppendorf micromanipulators on a Nikon Eclipse Ti-U inverted microscope. Oocytes were incubated overnight to allow for the expression of the fluorescent labels.We analyzed chromatin dynamics in live oocytes, using recombinant Histone H2B-RFP and a TALEN vector specific for centromeric major satellite sequences. Briefly, GV stage oocytes were microinjected with capped mRNAs encoding fluorescently labeled histone H2B-fusion proteins  as well as transcription activator-like effector (TALE)-mClover transcripts against repetitive major satellite genomic sequences (green) following vitro transcription from plasmids pGEMHE-H2B-RFP For the analysis of the effects of γ-irradiation, oocytes were washed and transferred to a micro drop of Embryomax M2 medium  supplemented with milrinone under mineral oil and subjected to a non-lethal dose of γ-irradiation  using a Mark I 68A Cesium-137 gamma irradiator . Oocytes were then transferred to a fresh micro drop of MEM/BSA medium supplemented with milrinone under mineral oil and cultured for 30 min, 5 h, or 24 h before fixing and immunochemistry as described above. Alternatively, chromatin domain movements were monitored in real time by live-cell imaging and confocal microscopy, followed by SR-SIM analysis as described below.2, 5% O2, and 90% N2. Oocytes were imaged using an Eclipse Ti-U/D-Eclipse C1 laser-scanning confocal microscope equipped with a ×40 objective lens following sequential (frame lambda) excitation of mClover/EGFP with a 488 nm and H2B-RFP with a 561 nm Coherent Sapphire laser. Image acquisition was conducted using EZC1 3.91 software (Nikon) with a step size of 2 μm and a Z-stack range of ~60 μm. Images were acquired every 5 min beginning at 30 min post irradiation for 5 h and subsequently analyzed by maximum intensity and 3D-reconstructions using NIH Elements 4.0 software and IMARIS . Heterochromatin domains labeled with TALE-mClover were tracked in 3D using the tracking module in NIS Elements applying identical thresholds on a Nikon Eclipse Ti-U/D-Eclipse C1 laser scanning confocal microscope equipped with a ×40 objective. Speed in µm/s and the total path length in µm were assessed for each chromocenter individually. Image acquisition was conducted using EZ-C1 software (Nikon) with a step size of 2 µm and a Z-stack range of 20 µm. Imaging data were subsequently analyzed by maximum intensity and 3D-reconstructions using NIH Elements software (Nikon).Live-cell-imaging was conducted by confocal microscopy on groups of irradiated and non-irradiated oocytes in micro drops of MEM/BSA medium supplemented with milrinone under mineral oil in an environmental chamber with an atmosphere of 5% CO15. Imaging was conducted using a Zeiss ELYRA S1 Superresolution system on an Axio Observer Z1 inverted microscope stand equipped with a 100X oil immersion lens and three laser lines: 405, 488, and 561 nm. Images were generated using 3 SIM rotations and individual Z-scans (0.091 μm) were processed with ZEN 2.3 software with a SIM analysis module to generate SR-SIM computations and maximum intensity projections at the Biomedical Microscopy Core (BMC) facility, University of Georgia. Quantitative analysis of chromocenter organization was conducted using Imaris x64 9.5.0 software (Bitplane). CZi files containing maximum intensity projections of the entire germinal vesicle were imported into Imaris. Pericentric heterochromatin blocks (chromocenters) were initially classified based on the DAPI fluorescence and major Satellite TALE-mClover channels using the automatic new surface function. Surface grain size was set at 0.0500 μm. The thresholds were 8700 for the absolute DAPI intensity and 2.30 μm3 for the volume so that DAPI intense heterochromatin blocks assembled as chromocenters could be automatically classified. 3D-surface-renderings of individual chromocenters as well as select chromatin fibers were automatically generated. We used the manual ‘wizard’ tool to draw the surface of the entire nucleus or germinal vesicle and the animation function of Imaris to generate a 3D-rendering rotating 720 degrees in the horizontal direction. The Vantage tab was used for quantitative analysis of chromocenter volume where the Y-axis displays chromocenter volume in μm3 and the X-axis indicates mean DAPI intensity. All epifluorescence images were processed using Photoshop 2.0 (Adobe) for linear adjustments and cropping of fluorescent images. No gamma adjustments were made.Oocyte chromatin was analyzed by high-resolution structured illumination microscopy (SR-SIM)n = 59) and γ-irradiated (n = 59) denuded oocytes were transferred to microcentrifuge tubes containing 3 μl of lysis buffer  and immediately frozen in liquid nitrogen. Analysis was conducted in two independent experimental replicates. Total RNA quality on each sample was determined using the Agilent 2100 Bioanalyzer system. Quality control prior to library construction included RNA concentration, an RNA integrity number of >8, 28/S/18S of >1.0 and fragment length distribution. Isolation of mRNA was conducted using oligo(dT). Library sequencing was performed using a Hiseq 4000 sequencing platform with paired-end 100 bp reads at the Beijing Genomics Institute. For each sample, >88 Million clean PE 100 reads or 8.93 Gb of data were obtained in FASTQ format. Quality-trimmed reads were aligned to the mouse genome reference GRCm38 using Bowtie2. Differential expression analysis between control and irradiated oocytes was conducted using the edgeR package. Transcripts with a log2-fold change of >2 and FDR < 0.05 were considered significant for differential expression (DEGs). DEGs were detected using DEseq2. For functional annotation, the gene ontology functional enrichment analysis and the Kyoto Encyplopedia of Genes and Genomes (KEGG) were used for DEGs classification using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE248416.Transcriptionally active NSN oocytes obtained on day 16 of post-natal development were processed for RNA-seq by first removing the zona pellucida with Tyrode’s solution (Sigma) and allowed to recover in MEM/BSA culture medium for 5 min. Control (n = 50) of denuded oocytes control and irradiated (24 h) oocytes using the miRNeasy kit (Qiagen) and subsequently reverse transcribed according to manufacturer’s instructions to assess transcript levels using validated RT2 qPCR Primer Assays  in two biological replicates. qPCR results were normalized against Gapdh  as a housekeeping control.For qPCR assays to validate differential expression following irradiation, mRNA was isolated from groups  algorithm of MultiTIMER35 was replaced by the gradient boosting machine (GBM) algorithm64, and the model was trained on 199 transcriptome profiles from the ArchS4 database of GV, control wild-type oocytes from mice of different ages. We also added the two control and two irradiated transcriptomes from our database into the training. To discover conserved cellular processes that are dysregulated in both aged and irradiated oocytes, the model was trained to predict the age of transcriptome profiles of non-irradiated oocytes accurately while also predicting advanced biological age for the transcriptome of irradiated oocytes. The h2o framework in R was used for machine learning applications65.We adapted the use of a multi-tissue RNA clock MultiTIMER36. These datasets all participated in the training of the model.To identify the most important cellular processes affected in aged and irradiated oocytes we compared the average normalized count value from two replicate transcriptomes from young non-irradiated oocytes and young irradiated oocytes from our study as well as the average normalized count value of transcriptomes from a published dataset of 14-month aged non-irradiated oocytes66. We define gene impact as the influence of a transcriptomic change of a gene in model prediction, and it is calculated by:I is the variable importance of the gene in model and D is the difference in normalized count value from baseline.We established our young control transcriptome as the baseline transcriptome. Then, in both young-irradiated and aged non-irradiated transcriptomes, we calculated the gene impact of all genes in the 29 top-level processes in the mouse version of the Molecular-Biology-of-the-Cell Ontology (process level 1)The sum of gene impact in each process is defined as the process impact, which indicates the level of impact on the model prediction exerted by the dysregulation of these conserved cellular processes. These process impact values were normalized to a range of 0–1 and drawn on a heatmap.P < 0.05 and P values are indicated. Box plots depict the median value as well as upper and lower quartiles with whiskers representing the minimum and maximum values observed. Dot plots show individual observations as dots as well as the mean with whiskers representing the standard deviation (s.d.).All data are presented as the mean values (±s.d.) from at least three independent experimental replicates unless stated otherwise. Comparison of all pairs was conducted using two-sided parametric and nonparametric tests (unpaired t-test or Mann–Whitney tests) according to the sample distribution (D’Agostino-Pearson) with GraphPad Prism 6 software or ggplot2 framework in R. Analysis of variance was conducted using one-way ANOVA with Tukey-s multiple comparison tests. Differences were considered significant when Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Reporting Summary


Brucella melitensis at a 1-kb resolution. We observed that the contact heat maps of the two B. melitensis chromosomes contain a prominent diagonal and a secondary diagonal. Then, 79 chromatin interaction domains (CIDs) were detected at an optical density at 600 nm (OD600) of 0.4 , with the longest CID being 106 kb and the shortest being 12 kb. Moreover, we obtained 49,363 significant cis-interaction loci and 59,953 significant trans-interaction loci. Meanwhile, 82 CIDs of B. melitensis at an OD600 of 1.5 (stationary phase) were detected, with the longest CID being 94 kb and the shortest being 16 kb. In addition, 25,965 significant cis-interaction loci and 35,938 significant trans-interaction loci were obtained in this phase. Furthermore, we found that as the B. melitensis cells grew from the logarithmic to the plateau phase, the frequency of short-range interactions increased, while that of long-range interactions decreased. Finally, combined analysis of 3D genome and whole-genome transcriptome (RNA-seq) data revealed that the strength of short-range interactions in Chr1 is specifically and strongly correlated with gene expression. Overall, our study provides a global view of the chromatin interactions in the B. melitensis chromosomes, which will serve as a resource for further study of the spatial regulation of gene expression in Brucella.The three-dimensional (3D) genome structure of an organism or cell is highly relevant to its biological activities, but the availability of 3D genome information for bacteria, especially intracellular pathogens, is still limited. Here, we used Hi-C (high-throughput chromosome conformation capture) technology to determine the 3D chromosome structures of exponential- and stationary-phase IMPORTANCE The spatial structure of chromatin plays important roles in normal cell functions and in the regulation of gene expression. Three-dimensional genome sequencing has been performed in many mammals and plants, but the availability of such data for bacteria, especially intracellular pathogens, is still limited. Approximately 10% of sequenced bacterial genomes contain more than one replicon. However, how multiple replicons are organized within bacterial cells, how they interact, and whether these interactions help to maintain or segregate these multipartite genomes are unresolved issues. Brucella is a Gram-negative, facultative intracellular, and zoonotic bacterium. Except for Brucella suis biovar 3, Brucella species have two chromosomes. Here, we applied Hi-C technology to determine the 3D genome structures of exponential- and stationary-phase Brucella melitensis chromosomes at a 1-kb resolution. Combined analysis of the 3D genome and RNA-seq data indicated that the strength of short-range interactions in B. melitensis Chr1 is specifically and strongly correlated with gene expression. Our study provides a resource to achieve a deeper understanding of the spatial regulation of gene expression in Brucella. Arabidopsis  (–Vibrio)  –28 and a–Vibrio) , 22 haveBrucella species are Gram-negative, facultative intracellular bacteria of the class Alphaproteobacteria. The pathogens can be transmitted from livestock and wildlife to humans via contact with infected animals or animal products through the respiratory, mucosal, or digestive tract. The clinical symptoms of brucellosis manifest as undulant fever, sweating, joint pain, splenomegaly, and infertility  and Chr2 are 2.12 Mb and 1.18 Mb, respectively. There are some differences in the types of genes on these two chromosomes  data revealed that the strength of short-range interactions in B. melitensis Chr1 is specifically and strongly correlated with gene expression.In this study, we applied Hi-C technology to determine the 3D genome structure of exponential- and stationary-phase Brucella, we applied Hi-C technology (600] of 0.4 [AOD0.4]) . A second, less prominent diagonal reflects lower-frequency interactions between loci on one arm of the chromosome and those on the opposite arm   and 1.5 (right)   and statAOD0.4])  B. melitactions) . Thus, tdiagonal  and B. Te genome . Accordianalyzed . The conB. melitensis at an OD600 of 0.4, 79 CID regions were detected, with the longest being 106 kb and the shortest being 12 kb. In B. melitensis at an OD600 of 1.5, 82 CID regions were detected, with the longest being 94 kb and the shortest being 16 kb (Table S1). This may suggest that as bacteria transition to stationary phase, the number of CIDs increases and the range becomes smaller. In order to compare the stability of the CID boundary structure between the two growth stages, we compared the CID boundaries between the samples according to the 2-kb resolution CID analysis in each sample and identified the CID boundaries that were shared between the samples and unique to each sample. Figure S1 shows that AOD0.4 (logarithmic-phase sample) shared 41 CIDs with AOD1.5 (plateau sample), while AOD0.4 and AOD1.5 had 36 and 39 unique CIDs, respectively. This result shows that the variation in CIDs during bacterial growth is relatively high.Chromatin interaction domains CIDs) refer to the high-level structures in genome space with relatively strong local interactions found on prokaryotic interaction maps . These as refer tB. melitensis were calculated. The results showed that there was no significant difference between the border and interior of the CIDs in terms of gene density distribution  or GC content distribution  at an OD600 of 0.4 or 1.5.CIDs are usually divided into border and interior regions. According to the results of CID analysis, the gene density distribution and GC content distribution of CID in In eukaryotes, TAD boundaries are enriched in highly expressed genes, and structural proteins such as CTCF (CCCTC-binding factor) and cohesin are usually enriched at TAD boundaries in the human genome . The chaB. melitensis were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis . GO analysis showed that the biological functions of the CID boundary genes were mainly enriched in metabolism and cellular processes, and their subcellular localization was mainly concentrated in the membrane and cell parts. Molecular functions were mainly enriched in catalytic activity, binding, and transport function . KEGG analysis showed that B. melitensis CID boundary genes were mainly involved in metabolism-related pathways . These results suggest that CID boundary genes may play important roles in B. melitensis growth and metabolism. Furthermore, to explore the functions of genes within the boundaries of the CIDs that were unique to each sample, the gene sets located within the boundaries of differential CIDs were functionally annotated . We also obtained 25,965 significant cis-interaction loci and 35,938 significant trans-interaction loci at the stable growth phase (Table S5). cis-interaction loci of B. melitensis at OD600s of 0.4 and 1.5. trans-interaction loci of B. melitensis at OD600s of 0.4 and 1.5.DNA loci that are distant within the genome may become spatially close via the formation of loops, bringing different biological domains closer, which impacts transcriptional regulation , 46. Fittrans-interactions in 3D structure, regulating the expression of different olfactory receptor genes . When the interaction distance was longer than 800 kb, the frequency of cis-interactions decreased. In addition, a comparison of Fig. S4A and B shows that as the bacteria grew from logarithmic to plateau phase, the proportion of short-range interactions (<200 kb) increased, while that of long-range interactions (>200 kb) decreased.Significant Brucella cultures more intuitively, an interaction subtraction matrix was utilized . Remarkably, these correlations were also observed in contact maps of E. coli and B. subtilis generated by different laboratories using different enzymes and cross-linking conditions  are a class of nucleic acid-binding basic proteins present in prokaryotic cells that can bind to bacterial chromosomes to form compact structures—that is, nucleoids. Local binding of NAP H-NS in ent ways . A recens of DNA . HoweverB. melitensis chromosomes at a 1-kb resolution. In total, 79 CID regions, 49,363 significant cis-interaction loci, and 59,953 significant trans-interaction loci were detected in B. melitensis at the logarithmic growth phase. Meanwhile, a total of 82 CID regions, 25,965 significant cis-interaction loci, and 35,938 significant trans-interaction loci were obtained for B. melitensis in the stable growth phase. Furthermore, combined analysis of 3D genome and RNA-seq data revealed that the strength of short-range interactions in B. melitensis Chr1 is specifically and strongly correlated with gene expression. Taken together, the results of our study provide a model to explain certain differences of Brucella in different growth periods from a 3D genome perspective and will serve as a resource for deeper study of the spatial regulation of gene expression in Brucella, which will contribute to further understanding of Brucella’s growth and pathogenesis.This study provides whole-genome contact heat maps of the exponential- and stationary-phase B. melitensis 16M was grown at 37°C in either tryptic soy broth (TSB)  or tryptic soy agar (TSA) . The experiments on B. melitensis were conducted in a biosafety level 3 laboratory.Bacterial culture was performed as reported previously , 52. BriB. melitensis cells were grown in TSB, and bacterial growth was measured via optical density at 600 nm (OD600). Bacterial cultures were collected when the OD600 value reached 0.4  or 1.5 (stationary phase). To fix the long-range DNA interactions, bacterial DNA was cross-linked with 3% formaldehyde  for 30 min at room temperature and another 30 min at 4°C. The reaction was stopped with 375 mM glycine for 15 min at 4°C, and the cells were washed with SET buffer  by centrifugation at 2,000 × g for 5 min at 4°C. The supernatant was discarded, and the cells were resuspended thoroughly in 3 mL SET buffer. Aliquots of bacteria were frozen at −80°C.Hi-C (chromosome conformation capture with deep sequencing) experiments were performed as described previously, with slight modifications , 14. B. Hi-C library construction and sequencing were carried out as described previously . BrieflyB. melitensis 16M was grown at 37°C and 180 rpm to an OD600 of 0.6 to ~0.8. The growth of the bacterial culture (5 mL per sample) was stopped by adding 0.625 mL of prechilled stop solution (5% phenol–95% ethanol). Cells were harvested by centrifugation for 10 min at 6,000 × g, and the resulting pellet was stored at −80°C. Frozen cell pellets were thawed on ice and resuspended in 100 μL per pellet of TE buffer . Total RNA was extracted using TRIzol reagent . The RNA samples were then subjected to treatment with DNase I  to remove genomic DNA. The concentration and quality of each sample were evaluated using a NanoDrop instrument  and agarose gel electrophoresis.RNA isolation was performed as described previously , 52. Briin vivo with RNAprotect bacterial reagent (Qiagen) and purified with an RNeasy Protect bacterial minikit (Qiagen). rRNA was depleted with a RiboZero-rRNA removal kit for bacteria (Epicenter). Libraries were prepared according to the instructions accompanying the Illumina TruSeq SBS Transcription kit.RNA library construction and sequencing were performed as described . BrieflyB. melitensis 16M strain, and valid paired reads were generated by the hiclib pipeline. The reliability of the Hi-C data from the two replicates was assessed with Genome DISCO. Chromatin interaction domains (CIDs) were identified by calculating the insulation score values using the software cworld-dekker. The intra- and interchromosomal interactions at the resolution of 1-kb bins were selected by Fit-Hi-C software  value of ≤0.01, and contact count of >2. Motif scanning was carried out in the JASPAR database (http://jaspar.genereg.net) using MEME Suite (http://meme-suite.org).The sequenced raw reads were filtered and quality evaluated using Trimmomatic  and Fastsoftware , with thB. melitensis genome was predicted from Hi-C data by the PASTIS software using a 1-kb resolution matrix with the multidimensional scaling (MDS) module  module . In the software .B. melitensis 16M strain generated in this study have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession no. PRJNA918778 and PRJNA916855, respectively.The Hi-C and RNA-seq data of the


T. brucei), in RNA polymerase II termination sites and sub-telomeric regions . This hypermodified nucleotide is synthesized in two steps with the participation of two distinct thymidine hydroxylases, J-binding protein 1 and 2  and a β-glucosyl transferase. A third J-binding protein, named JBP3, was recently identified as part of a multimeric complex. Although its structural similarities with JBP1, it seems not to be involved in J biosynthesis but to play roles in gene expression regulation in trypanosomatids. Over the years, with the characterization of JBP1 and JBP2 mutant lines, Base J functions have been targeted and shone a light on that matter, showing genus-specific features. This review aims to explore Base J’s reported participation as a regulator of RNA polymerase II transcription termination and to summarize the functional and structural characteristics and similarities of the remarkable JBP proteins in pathogenic trypanosomatids.β-D-glucopyranosyloxymethiluracil (Base J) is a modified thymidine base found in kinetoplastids and some related organisms. Interestingly, Base J distribution into the genome can vary depending on the organism and its life stage. Base J is reported to be found mostly at telomeric repeats, on inactive variant surface glycoproteins (VSG’s) expression sites (e.g.,  Trypanosoma and the Leishmania genera, which possess dixenous development. Some of these trypanosomatids are pathogens of medical importance, causing diseases of a range of severity whose treatment and control methods are still precarious, urging the finding of new anti-parasitic drug targets [Leishmania sp. up to 55 Mb in Trypanosoma cruzi. Most of their genes are organized in large clusters, polycistronicaly transcribed and processed by trans-splicing [Trypanosomatids comprise a diverse group of protozoan parasites of the class Kinetoplastids, among which are species of the  targets . Trypanosplicing ,3,4.Trypanosoma brucei and T. cruzi present a set of genes involved with virulence being transcribed at the subtelomeric position [T. brucei) and towards the end of the chromosome are the telomeres. They are arrangements of DNA and proteins crucial to cell cycle maintenance and important cellular processes such as cell aging, genome integrity, and nuclear arrangement [Moreover, many aspects of transcriptional regulation are still under investigation, mainly due to the absence of canonical promoters in RNA polymerase II transcribed genes. Species such as position ,6, transangement ,8. Moreoangement .Trypanosoma brucei [Bernards and collaborators first reported a modified nucleotide at the telomeres on trypanosomatids and closely related organisms. This nucleotide, later known as Base J, was thought to affect antigenic variation in the bloodstream form of a brucei . Later, a brucei ,12, openBase J, or β-D-glucopyranosyloxymethiluracil, is a modified thymidine whose synthesis is divided into two steps. In the first step, two thymidine hydroxylases, either J-binding protein 1 (JBP1) or J-binding protein 2 (JBP2), at a specific location on the nuclear DNA, alter a thymidine into hydroxymethyldeoxyuridine (HOMedU). In the second step, the HOMedU is glycosylated by a β-glucosyl transferase and transformed into Base J . T. brucei, Base J was first observed at inactive VSG expression sites (ES) only in the bloodstream form of the parasite [T. brucei. About 13% of thymidine was modified into Base J at purified telomeric repeats, whereas only 0.8% was in total DNA. In addition, in bloodstream forms, approximately 50% of Base J was estimated to be located at telomeric repeats [T. brucei [T. brucei and a histone variant, H3.V, which regulates transcription termination [T. brucei [T. brucei, PJW/PP1 complex regulates termination through JBP3–Base J interactions and dephosphorylation of proteins such as RNA polymerase II and termination factors via PP1 [In parasite . It replparasite ,16. Diff repeats . Localiz repeats . Later,  repeats . However. brucei . Yet, thmination , indicatmination . Another. brucei . Later, . brucei . This co via PP1 . DisruptL. tarentolae extracts [Leishmania uses Base J and proteins involved in chromatin remodeling and transcriptional regulation to induce RNA polymerase II transcription termination. Furthermore, the authors showed that the ablation of JBP3 in L. tarentolae resulted in a substantial increase in transcriptional readthrough at the 3′ end of most PTUs. These results suggest that JBP3 might recruit one or more of these chromatin remodeling complexes to the J-containing regions at the end of PTUs, which halts the progression of RNA polymerase II transcriptional activity.Similarly, Jensen and collaborators identified JBP3 in extracts . They deT. cruzi, it is found in all parasite life stages, with it being upregulated (~two fold) in the infective mammalian stage [T. cruzi, like other trypanosomatids, shuttles between the mammalian (metacyclic trypomastigotes and amastigotes) and the insect/vector (epimastigotes) forms. Around a quarter of T. cruzi’s Base J content is localized at subtelomeric regions, which is enriched in life-stage-specific surface glycoprotein genes involved in the parasite’s virulence [T. cruzi. The knocking out of the two enzymes that regulate Base J synthesis (JBP1 and JBP2) decreased Base J levels at transcription initiation sites, correlating with aincreased RNA polymerase II transcription and a genome-wide increase in gene expression. Therefore, in T. cruzi, Base J may act as an epigenetic factor regulating RNA polymerase II transcription [Although Base J is also primarily a modification found at the chromosome ends, in an stage . T. cruzirulence . Moreovecription .Leishmania genus present an interesting Base J occurrence and distribution profile. Prior investigations showed that 99% of all Base J that co-migrated with telomeric repeat-containing DNA in J-immunoblots and Southern blots of fragmented DNA were all Base J content localized at the telomeric regions. The meaning is that 99% of Base J in Leishmania should be located at parasite telomeres [T. brucei can survive without Base J [Leishmania tarentolae, for example, seems to require Base J to survive since JBP1 knockout (KO) cells are not viable [Leishmania telomeres is maintaining cell homeostasis, although no detectable telomeric phenotypes were described in parasites maintained in early passages [Leishmania. They showed that out of the telomeres in L. major and L. tarentolae, the remaining Base J content had been confined at RNA polymerase II transcription termination sites [L. tarentolae JBP2 KO cells submitted to BrdU treatment died due to the overall Base J reduction over these genetic and experimental conditions. In addition, they also showed that the loss of Base J is accompanied by massive readthrough of normal RNA polymerase II transcriptional termination sites. Therefore, it is likely that Base J is required for proper transcription termination and that the absence of internal Base J is lethal for L. tarentolae due to the massive readthrough of transcriptional stops [Moreover, parasites belonging to the elomeres . Whereast Base J ,28, Leist viable . In contt viable . Interest viable . Therefopassages . Van Leeon sites . Thus, ial stops .L. major, although the reduction of Base J by dimethyloxalylglycine (DMOG) also resulted in genome-wide transcriptional readthrough at convergent strand switch regions (cSSRs) and head–tail (HT) sites, it does not trigger cell death [L. major, Base J regulates RNA polymerase II transcriptional termination at the end of each PTU, preventing the generation of genome-wide antisense RNAs. In contrast, in ll death . Thus, iThe presence of Base J only at specific sites replacing thymidine residues in the trypanosomatids genome was considered the first indication that Base J is a modified nucleotide and not a randomly incorporated mutated deoxynucleotide during DNA synthesis . TherefoT. brucei bloodstream forms, showing a specific binding activity to Base-J-containing duplex DNA. Similar results were also obtained with extracts of the non-pathogenic Crithidia fasciculata and L. tarentolae. In addition, recombinant JBP1 expressed in Escherichia coli also showed its specificity to bind Base J DNA content [JBP1 was first identified in nuclear extracts of  content . T. brucei using JBP1 KO lines indicated that the disruption of JBP1 does not affect growth, gene expression, or the stability of some repetitive DNA sequences [T. brucei DNA by offering the cells the Base J precursor HOMedU, is lost by simple dilution upon cell duplication [Sabatini and collaborators further explored JBP1 biochemical characteristics using J-DNA substrates and purified recombinant JBP1 protein . They shequences . Howeverlication , indicatT. brucei bloodstream form cells and interacts with nuclear chromatin. The expression of JBP2 in T. brucei procyclic cells leads to de novo Base J synthesis within telomeric regions. However, this activity is inhibited upon mutagenesis of conserved residues critical for SWI2/SNF2 function [T. brucei bloodstream forms resulted in a decrease of five-fold in Base J levels and the inability of the parasites to stimulate Base J synthesis de novo in newly generated telomeric arrays [Since JBP1 was shown to be a Base-J-specific DNA binding protein, the question of how the de novo synthesis of Base J occurs remained unanswered. DiPaolo and collaborators identified the homolog of JBP1, JBP2, which contains a domain related to the SWI2/SNF2 family of chromatin remodeling proteins, which is upregulated in function . Consistc arrays .2+ and 2-oxoglutarate-dependent dioxygenases. In addition, the replacement of conserved residues putatively involved in Fe2+ and 2-oxoglutarate-binding inactivates the ability of JBP1 and JBP2 to contribute to Base J synthesis without affecting its ability to bind to J-DNA [Additionally, Yu and collaborators and Cliffe and collaborators showed that JBP1 and JBP2 belong to the family of Feto J-DNA . Thus, ato J-DNA ,34,38  and AlphaFold . Unfortunately, no solved in vitro 3D structures are available for the full-length JBP1 or JBP2 proteins, but there are two entries at the Protein Data Bank (PDB: 8BBM and 2XSE) [L. tarentolae JBP1 J-DNA-binding domain (J-DBD) obtained through X-ray diffraction [The in silico structure of JBP1 and JBP2 can be found in protein databases such as UniProt (nd 2XSE) . Both enfraction ,44. In cRepresentative structures for the three JBPs obtained through AlphaFold and UniProt are shown in 2+/2-oxoglutarate-dependent dioxygenases [2+ binding, and an arginine important for binding 2-oxoglutarate  by Kieft and collaborators using comparative modeling and alignment [L. major. The MYND domain encompasses cysteine and histidine residues organized like fingers to form binding sites for Zinc or other metals [Leishmania’s JBP3 MYND domain is serine-rich, thus requiring further investigations.The structure of the JBP3 protein started to be characterized in lignment . They der metals . DiffereLeishmania  but a comparatively lower degree of conservation among the Trypanosoma species . This difference in conservation may indicate a higher selective pressure to preserve the primary structure of proteins in Leishmania than in Trypanosoma.We could not find information regarding the conservation and the comparative structural aspects of the JBPs over trypanosomatids species. Therefore, using public information on JBPs sequences, we performed amino acid sequence alignments among the three main human pathogenic species C using BT. brucei can survive without Base J, Leishmania spp. seems to require Base J to survive. Base J synthesis involves hydroxylation and further glycosylation of thymidine residues. The first step of Base J biosynthesis is catalyzed by two thymidine hydroxylases, JBP1 and JBP2, members of the Fe2+- and 2-oxoglutarate-dependent oxygenase family. While JBP1 is a J-DNA-binding protein  mediating the propagation/maintenance of Base J in the genome, JBP2 appears to be mainly responsible for de novo and site-specific Base J synthesis. The JBP2 SWI2/SNF2-like domain, in turn, recognizes and binds to certain chromosome regions, hydrolyzes ATP, and allows the J-synthesis machinery to gain access to DNA by chromatin remodeling.Base J is a unique and relevant modified nucleotide found in trypanosomatids. It is localized at subtelomeric and telomeric repeats and RNA polymerase II termination sites. The importance of Base J in different species of trypanosomatids has been accessed by investigating phenotypes of knockout lines for the enzymes producing Base J and by preventing Base J biosynthesis. For example, whereas Leishmania genus. An additional J-binding protein was recently described in T. brucei and L. tarentolae, the JBP3. Although unrelated to Base J biosynthesis, JBP3 regulates gene expression in trypanosomatids by binding J-DNA with a multimeric protein complex involved with chromatin remodeling.Regarding their structure, JBP1 and JBP2 are considerably conserved, especially within the i) how specific thymine residues are targeted for J-modification. Would that be a random mechanism, or do unknown protein factors control it during development or environmental changes? (ii) Would the mechanism of J regulating transcription termination be genus- or species-specific? Is there a connection with parasite virulence/pathogenicity? (iii) Is the massive presence of Base J at L. major telomeres indeed involved with the transcriptional regulation of telomeric lncRNA such as TERRA? [iv) What is the function of the antisense transcripts detected from regions upstream of transcription initiate sites? Are they only the result of bi-directional transcription activity? Which factors control their degradation since they are rapidly degraded and very hard to be detected? (v) If JBP complexes are similar in composition to TETs in higher eukaryotes, why do they differ in composition between L. tarentolae and T. brucei? Is that a rule depending on the genus and parasite species? Finally, how important are these findings concerning base J as an epigenetic factor?Much has been done to uncover Base J’s relevance in trypanosomatids gene regulation. Nevertheless, much needs to be done to answer important questions. Amongst them, we can mention (s TERRA?  (iv) WhaLeishmania species suggests that JBPs may play extra roles in parasite biology. Therefore, those mentioned above and many other unanswered questions deserve more attention from the parasitology community.The present review aimed not only to show the state of the art of our knowledge of Base J and J-proteins’ functions but to show that much has yet to be accomplished in the field. Base J is one of the few known epigenetic factors controlling gene expression in some trypanosomatids. Its absence in some


The interplay between genetic alterations and metabolic dysregulation is increasingly recognized as a pivotal axis in cancer pathogenesis. Both elements are mutually reinforcing, thereby expediting the ontogeny and progression of malignant neoplasms. Intriguingly, recent findings have highlighted the translocation of metabolites and metabolic enzymes from the cytoplasm into the nuclear compartment, where they appear to be intimately associated with tumor cell proliferation. Despite these advancements, significant gaps persist in our understanding of their specific roles within the nuclear milieu, their modulatory effects on gene transcription and cellular proliferation, and the intricacies of their coordination with the genomic landscape. In this comprehensive review, we endeavor to elucidate the regulatory landscape of metabolic signaling within the nuclear domain, namely nuclear metabolic signaling involving metabolites and metabolic enzymes. We explore the roles and molecular mechanisms through which metabolic flux and enzymatic activity impact critical nuclear processes, including epigenetic modulation, DNA damage repair, and gene expression regulation. In conclusion, we underscore the paramount significance of nuclear metabolic signaling in cancer biology and enumerate potential therapeutic targets, associated pharmacological interventions, and implications for clinical applications. Importantly, these emergent findings not only augment our conceptual understanding of tumoral metabolism but also herald the potential for innovative therapeutic paradigms targeting the metabolism–genome transcriptional axis. 1. Cell metabolism and gene control are significantly integrated and regulated by one another.2. There is a positive feedback loop of metabolism‐epigenetics‐gene regulation which control cancer progression.3. The discovery of metabolic enzymes and their functions in the nucleus will provide more insights into cancer therapy. The crosstalk between these two domains is an imperative subject that holds significant implications for both our understanding of cancer progression and therapeutic innovation. In eukaryotic cells, genetic information is primarily housed within chromatin structures, whose integrity is governed by complex processes such as DNA methylation and an array of histone modifications.O‐GlcNAcylation, have been documented on both histone amino‐terminal tails and globular histone cores.Chromatin architecture is orchestrated through the strategic assembly of nucleosomes, which are composed of histone octamers—formed by histone proteins H2A, H2B, H3, and H4—around which superhelical DNA is wrapped.Metabolic reprogramming constitutes a hallmark of cancer pathobiology, significantly influencing a diverse range of abnormal metabolic pathways, including glycolysis, lipogenesis, and amino acid biosynthesis and catabolism, all of which have ramifications for cancer progression and treatment responsiveness.Elucidating the nuclear functions of metabolic signaling—including both metabolites and metabolic enzymes, collectively termed as “nuclear metabolic signaling”—promises not only to unveil novel therapeutic targets but also to inspire innovative treatment strategies targeting the dual‐function metabolic enzymes or the intricate metabolism‐gene transcription axis. These potential targets are implicated not merely in cellular metabolism but also in an array of nuclear processes that influence tumoral proliferation, such as gene transcription, DNA damage repair, and chromatin modification. Hence, the identification and characterization of these nuclear roles are of paramount importance for advancing cancer research and therapeutic modalities.In this review, we aim to provide a comprehensive analysis of metabolic signaling within the nuclear compartment. We will examine the multifaceted functions of these signaling components in the nuclear environment, their impact on other nuclear biological processes, and the ensuing therapeutic strategies and clinical applications.2Metabolic reprogramming is increasingly acknowledged as a salient hallmark of cancer pathobiology. Such reprogramming culminates in an overabundance of metabolites and metabolic enzymes, which serve not only as nutritional substrates but also infiltrate the nuclear compartment to modulate gene expression, thereby fueling cancer cell proliferation. Consequently, elucidating the intricacies of nuclear metabolic signaling and its regulatory mechanisms has become an imperative avenue of inquiry. We will introduce roles of nuclear metabolism in cancer growth and mechanisms of nuclear metabolic dysregulation in following discussion. It includes various modifications which affect the epigenetic characteristics and nuclear metabolic enzymes which usually present in the cytoplasm.2.1The pathogenesis of cancer is an intricate tapestry woven from multiple contributing factors, among which metabolic anomalies stand as pivotal elements. Within this context, nuclear metabolic signaling represents an indispensable component of cellular metabolism, orchestrating a myriad of cellular functions essential for growth and proliferation. Metabolites that encroach upon the nuclear domain in tumor cells serve multifaceted roles—acting as cofactors, molecular donors for modifications, or agents that either potentiate or attenuate biochemical activities, thereby exerting a profound influence on the epigenetic framework.+) play regulatory roles in histone acetylation, thereby adding nuanced layers to an already complex epigenetic landscape.For instance, the S‐adenosylmethionine (SAM) to S‐adenosylhomocysteine (SAH) ratio serves as a crucial regulatory axis, modulating the activities of an array of methyltransferases, and thereby overseeing methylation patterns across DNA, RNA, and histones.+, ATP, and succinate. Alterations in the patterns of histone or DNA/RNA modification are frequently correlated with oncogenesis. Hence, nuclear metabolic signaling plays an instrumental role in the promotion of cancer cell growth and proliferation by modulating an array of oncogenes. Comprehensive insights into the role of nuclear metabolism could pave the way for the development of innovative and more efficacious therapeutic paradigms, thereby enhancing the landscape of cancer treatment.Significantly, these events are metabolically driven, requiring substrates or cofactors such as SAM, acetyl coenzyme A, α‐KG, NAD2.2Nuclear metabolic signaling orchestrates gene expression by influencing a spectrum of molecular mechanisms, encompassing epigenetic modifications, RNA/protein posttranslational modifications, and the moonlighting functions of metabolic enzymes within the nuclear milieu. These represent the predominant mechanisms through which nuclear metabolic signaling exerts its regulatory influence on gene expression.2.2.1Histone methylation, a prevalent posttranslational modification regulated by histone methylases (HMTs), is ubiquitously identified across mammalian species and demonstrates a significant association with oncogenesis. This modification principally involves the methylation of lysine (K) or arginine (R) residues, predominantly localized at the amino termini of core histones H3 and H4, including but not limited to the loci H3K4, H3K9, H3K36, H3K79, and H4K20.SAM serves as the universal methyl donor, predominantly synthesized from methionine via the one‐carbon metabolism pathway, facilitated by methionine adenosyl transferases.It is of considerable note that deletions involving methylthioadenosine phosphorylase (MTAP)—a pivotal enzyme in the methionine salvage pathway—have been implicated in multiple cancer phenotypes.Defects in serine metabolism exert a consequential impact on the levels of SAM in neoplastic cells. Serine serves as a principal donor of one‐carbon units to the folate cycle, an integral component of one‐carbon metabolismHistone demethylases (HDMs), colloquially termed “erasers,” possess the capability to expunge methylation moieties from histones. Importantly, these enzymes leverage a variety of metabolites as cofactors in the aforementioned biological cascade Figure . Two priThe mammalian genome is characterized by a high prevalence of CpG DNA methylation, with approximately 60−80% of CpG dinucleotides being methylated, excluding CpG islands—CpG‐rich domains that are predominantly unmethylated and encapsulate the promoters for up to 60% of genes.2+, O2, and α‐KG serve as pivotal cofactors in TET‐mediated reactionsDNA methylation represents a dynamic equilibrium between two antithetical processes: DNA methylation and DNA demethylation. The ten‐eleven translocation (TET) family of proteins and thymine DNA glycosylase function as catalytic facilitators of DNA demethylation.Histone acetylation constitutes a ubiquitous, reversible posttranslational modification mediated by two distinct classes of enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs) Figure . HATs orThe rate of enzymatic acetylation and deacetylation is contingent upon the availability of acyl group donors. Acetyl‐coenzyme A (acetyl‐CoA) serves as a principal substrate for the acetylation catalyzed by HATs Figure . The met2+‐responsive mechanism involving the NFAT1 transcription factor, acetyl‐CoA has been observed to promote glioblastoma cell migration and adhesion to the extracellular matrix.2+ levels, thereby facilitating the nuclear translocation of Ca2+‐dependent NFAT1. The transfer of acetyl‐CoA from the mitochondrial matrix to the cytosol is mediated by citrate, which is subsequently cleaved back into oxaloacetate and acetyl‐CoA by ACLY.In a CaNotably, histone acetylation serves as a critical facilitator of proficient DNA repair mechanisms. It aids in the recruitment of specialized repair proteins and ensures unimpeded access to double‐strand break (DSB) loci for the DNA repair machinery.Recent investigations have elucidated those posttranslational modifications of specific nuclear genes, including but not limited to ACSS, ACLY or ACLA, and PDC, exhibit a nuanced interplay with HATs or nuclear transcription factors, thus potentially influencing chromatin regulationACLY serves as a unique substrate that is sensitive to mechanistic target of rapamycin complex 2 (mTORC2), which subsequently modulates ACLY activity to enhance ACSS2‐mediated synthesis of acetyl‐CoA from acetate, thereby augmenting histone acetylation.Furthermore, ACSS2 plays a discernible role in furnishing acetyl‐CoA requisite for nuclear histone acetylation.Moreover, in the realm of prostate cancer, nuclear PDC has been shown to induce acetylation at histone 3 lysine 9 (H3K9) in the promoter regions of lipid‐associated genes, such as ACLY and squalene epoxidase, thereby facilitating lipogenesis and cellular proliferation.+ modulate and amplify the enzymatic activity of the sirtuin family, counteracting the inhibitory effect of nicotinamide (NAM), a precursor to NAD+.HDACs are zinc ion‐dependent enzymes and display subtype‐specific roles across a plethora of tumors. Elevated levels of HDAC1 have been reported in gastric and prostate cancers, while HDAC2 expression is abundant in colon, cervical, and gastric cancers.N‐ and O‐linked glycosylation is uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc).O‐GlcNAcylation process specifically entails the singular O‐linked attachment of GlcNAc moieties to serine and threonine residues on proteins. This dynamic posttranslational modification is orchestrated by two pivotal enzymes: O‐GlcNAc transferase (OGT) and O‐GlcNAcase, which catalyze the addition and removal of O‐GlcNAc modifications, respectively.Nucleotide sugars, which function as substrates for glycosylation processes, are generated through the hexosamine biosynthetic pathway (HBP). A salient substrate implicated in both Dysregulated O‐GlcNAcylation has been implicated in the modulation of signal transduction pathways, gene expression profiles, and metabolic processes, cumulatively contributing to oncogenic transformation and cancer progression.In specific cancer types such as breast and colon cancer, the knockdown of OGT engenders a significant diminution in H3K27me3 levels and in the protein stability of Enhancer of Zeste Homolog 2 (EZH2), without influencing the concentrations of H3K27 demethylases, UTX, and JMJD3.OGT itself is subject to direct phosphorylation at the Thr 444 site by AMPK. This phosphorylation disrupts OGT's chromatin interaction and attenuates histone H2B O‐GlcNAcylation, albeit without exerting a direct influence on OGT's enzymatic capabilities.Beyond histones, a repertoire of epigenetic regulators are also susceptible to O‐GlcNAcylation. For instance, OGT forms a complex with the methylcytosine dioxygenases TET1, TET2, and TET3—each of which is highly O‐GlcNAcylated. In a reciprocal fashion, these TET proteins anchor OGT to chromatin, where they regulate the conversion of 5mC to 5hmC, a process intricately modulated by both OGT and O‐GlcNAcylated TET proteins.Lipid moieties, characterized by their hydrophobic groups, can be covalently affixed to proteins via an array of linkages, including amide, ester, thioester, or thioether bonds. Such lipid‐modified proteins fulfill multifaceted roles, encompassing the regulation of protein functionality, subcellular localization, and intricate interactions with other cellular entities, such as proteins, lipids, cofactors, and nucleic acids.The cell surface receptor interferon alpha receptor 1 (IFNAR1) serves as a ligand for type I interferons (IFNs), thereby facilitating the activation of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) to modulate the transcription of downstream genes via IFN‐stimulated response elements Figures . HoweverOct4 (POU5F1), a transcription factor, exists in variants including Oct4A, Oct4B, and Oct4B1.EZH2, a lysine methyltransferase, orchestrates DNA methylation to repress gene transcription.Wilms' tumor 1 protein undergoes a functional transformation from a transcriptional activator to a repressor via its interaction with the transcriptional corepressor BASP1.The burgeoning advances in RNA research have culminated in the identification of an expansive repertoire of RNA modifications, enabled by the advent of highly specialized, quantitatively accurate, and sensitively precise investigative methodologies. To this point, an array of over 170 discrete modifications have been meticulously characterized within various RNA classes, including tRNAs, ribosomal RNAs (rRNAs), long noncoding RNAs (lncRNAs), mRNAs, microRNAs (miRNAs), among others.l‐cysteine hydropersulfide (LCYH), SAM, SAH, GSH, and l‐methionine in tumor tissues. This diminution subsequently resulted in a significant decrement in m6A methylation and expression of immune checkpoints, including PD‐L1 and V‐domain Ig suppressor of T cell activation (VISTA).As a pivotal regulatory modality, RNA methylation exerts influence over an assortment of metabolic pathways, thereby shaping the trajectory of tumor initiation and subsequent progression. Recent scholarly investigations suggest that nuclear metabolic signaling has the potential to fine‐tune RNA methylation levels through a variety of mechanistic avenues, offering profound implications for the dynamics of cancer development. Analogous to DNA or histone methylation, the methyl groups pertinent to RNA methylation are derived from SAM, implicating that both the methionine and folate cycles exert an intricate regulatory influence on RNA methylation levels.Apart from the methionine and folate cycles, additional metabolic pathways and metabolites also hold the capacity to modulate m6A modifications. Under hypoxic conditions, HIF‐1α governs the transcription of YTHDF1, a recognized m6A reader, by directly engaging with its promoter region. Subsequently, YTHDF1 associates with m6A‐modified ATG2A and ATG14 mRNA, thereby facilitating the translational processes of autophagy‐related genes ATG2A and ATG14. This intricate cascade amplifies the autophagic process and contributes significantly to the pathogenesis of autophagy‐associated malignancies.Recent investigations have illuminated that nicotinamide adenine dinucleotide phosphate (NADP) and NAD are also instrumental in modulating mRNA m6A modification. Specifically, NADP serves to potentiate the enzymatic activity of the RNA demethylase FTO, thereby fine‐tuning mRNA m6A methylation profiles, which subsequently wield an influence on adipocyte differentiation during the adipogenesis process. This insight delineates a direct nexus between cellular metabolic processes and the intricate regulatory mechanisms of RNA m6A methylation.Moreover, the accretion of lactate within the tumor microenvironment has been shown to instigate histone H3K18 lactylation, engendering elevated expression of Mettl3 and m6A modification of Jak1 mRNA within tumor‐infiltrating myeloid cells. This sequential cascade amplifies the translation of Jak1 mRNA and fosters the phosphorylation of STAT3, thereby facilitating immune‐suppressive functionalities in these myeloid cells and mediating mechanisms for tumor immune evasion.In addition to m6A modifications, other methylation variances are also subject to modulation by metabolic pathways or specific metabolites. For instance, upon the assimilation of fatty acids derived from omental adipocytes, gastric cancer cells experience an upregulation in the transcription factor E2F1. This elevation subsequently propels the expression of m5C methyltransferase NSUN2 via cis‐regulatory mechanisms.Currently, burgeoning empirical evidence is increasingly establishing a compelling correlation between dysregulated RNA methylation and perturbed tumor metabolism, thereby contributing to the complexity of tumor progression. The frontier of anticancer therapeutics is being reshaped by an emergent focus on developing drugs targeting RNA methylation Table . NonethePhospholipids constitute a pivotal component of cellular membranes. While the primary understanding of phospholipids emanates from their salient biological functions within these membranes, it should be acknowledged that phospholipids and their derivatives, namely inositol phosphates (IPs), are ubiquitously present in the cellular nucleus and fulfill consequential biological roles therein.Phosphatidylethanolamine (PE) methylation serves as a paramount consumer of SAM. This methylation event participates in catalyzing the transsulfuration pathways imperative for the biosynthesis of cysteine and glutathione (GSH). Diminished PE methylation culminates in the intracellular accretion of SAM, thereby engendering hypermethylation of both histones and the key phosphatase PP2A. In the absence of PE methylation, particular loci on histones function as methyl sinks, catalyzing the conversion of SAM to SAH.Phosphatidylinositol 4,5‐bisphosphate (PIP2), a derivative of inositol phosphoric acid, engages in complex interactions with UBF, RNA polymerase I, and prefibrillarin, thus modulating the transcriptional activity of RNA polymerase I and nucleolar RNA processing.Collectively, these studies elucidate that aberrant nuclear lipid metabolism is intrinsically entangled with epigenomic regulation and may serve as a contributory factor in tumorigenesis. Despite the valuable insights garnered, our comprehension of nuclear lipid metabolism remains conspicuously incomplete. Additional research is imperative for elucidating these multifaceted molecular mechanisms, and may proffer innovative avenues for targeted cancer therapeutics.2.2.2Emerging evidence posits that a multitude of metabolic enzymes, typically confined to the cytoplasm or mitochondria, can translocate into the nucleus and exercise regulatory influence over gene transcription by altering chromatin conformation. In addition to this epigenetic role, these enzymes execute additional nuclear functions that encompass gene transcription, DNA damage repair, and posttranslational modifications of proteins Figure . These mRecent investigations have elucidated that hexokinase 2 (HK2), an enzyme belonging to the transferase kinase category that phosphorylates glucose to glucose‐6‐phosphate, is translocated to the nucleus in direct association with the DNA‐binding transcriptional repressor Mig1. This translocation is modulated by glucose concentration and is under the regulatory influence of phosphorylation by Snf1 kinase and dephosphorylation by the Glc7‐Reg1 phosphatase complex.Moreover, HK2 contains highly conserved cysteine residues that are sensitive to redox modification in response to reactive oxygen species (ROS) stimulation. For example, dehydroascorbic acid (DHA) forms a covalent bond with the active cysteine site of HK1, leading to irreversible enzymatic inactivity.Benitrobenrazide, an innovative selective HK2 inhibitor, demonstrates the capability to target the binding site and preclude glucose association with HK2, thus efficaciously inhibiting pancreatic cancer growth by disrupting glycolytic pathways.Within the glycolytic cascade, phosphofructokinase 1 (PFK1) functions as a rate‐limiting enzyme, facilitating the conversion of fructose 6‐phosphate to fructose 1,6‐bisphosphate. Various tetrameric isozymic variants of PFK1 coalesce to produce three distinct subunit types—namely, PFKM (muscle), PFKL (liver), and PFKP (platelet).Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) plays an instrumental role in the glycolytic pathway, catalyzing the conversion of glyceraldehyde 3‐phosphate to 1,3‐bisphosphoglycerate. Remarkably, it also exhibits a proclivity for direct interaction with the substrates of the G1/S‐specific cyclin E‐CDK2 complex, specifically OCT1 and NPAT, thereby orchestrating the transcriptional regulation of histone genes  during the initiation of apoptosis. This modification facilitates GAPDH's binding to Siah1, an E3 ubiquitin ligase, and culminates in its nuclear translocation, ultimately inducing cellular apoptosis. Importantly, S‐nitrosylation accentuates GAPDH's interaction with Siah1, whose nuclear localization signal acts as a catalyst for GAPDH's nuclear translocation.In glucose‐deprived conditions, cytoplasmic GAPDH is phosphorylated at the Ser122 site by activated AMPK. This phosphorylation serves as an impetus for GAPDH's nuclear translocation. Once localized to the nucleus, GAPDH engages in direct interactions with Sirt1, effectively displacing its repressor and activating the enzyme.Ceramide exerts a regulatory influence over GAPDH's nuclear localization in lung cancer cells through a cell cycle‐dependent mechanism, in addition to inhibiting its telomere‐binding function. Intriguingly, a rapid telomeric shortening is observed upon the inhibition of GAPDH expression, highlighting the critical role that nuclear GAPDH plays in telomere maintenance and protection.Under conditions of oxidative stress, cysteine 152 (Cys152), a conserved active site within GAPDH, undergoes posttranslational modifications via sulfonation or glutathionylation. Such redox‐mediated modifications possess the capacity to attenuate GAPDH enzymatic activity, thereby rerouting metabolic flux away from glycolysis toward the pentose phosphate pathway (PPP).High‐dose vitamin C therapy proves efficacious in mitigating the extensive depletion of GSH as well as the accumulation of ROS, thereby expediting the glutathionylation of GAPDH at the Cys152 site. Such modulation effectively inactivates GAPDH and exerts inhibitory effects on colon cancer cells harboring KRAS and BRAF mutations, primarily through the obstruction of glycolytic pathways.d‐glucopyranose has been reported to compete with NAD+ and inorganic phosphate (Pi) to impede GAPDH activity; however, its inhibitory efficacy in tumor models remains to be empirically validatedIn a therapeutic context, selegiline—a pharmacological agent employed in Parkinson's disease treatment—can inhibit the S‐nitrosylation of GAPDH, thereby precluding its interaction with Siah1 and constraining the nuclear translocation of the GAPDH‐Siah complex, thus limiting cellular apoptosis.N6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphos‐phatase 4 (PFKFB4) serves as an indispensable catalyst in the glycolytic pathway and is manifestly overexpressed in neoplastic cellsSRC‐3 undergoes specific phosphorylation at the serine 857 residue, mediated by the protein kinase activity of PFKFB4. Once phosphorylated, SRC‐3 engages in intricate interactions with ATF4 at the gene promoter locales, thereby engendering an upsurge in the expression of an array of metabolic enzymes. Noteworthy among these are transketolase, xanthine dehydrogenase, and adenosine monophosphate deaminase‐1.In the realm of therapeutic interventions, 5‐(N‐(8‐methoxy‐4‐quinolyl)amino)pentyl nitrate (5MPN) has been unequivocally identified as a potent, orally bioavailable, and selective inhibitor of PFKFB4. 5MPN operates as a competitive antagonist at the F6P binding site, consequently exerting a targeted inhibition on glucose metabolism. This inhibition effectually constrains the proliferative trajectory of a diverse range of malignanciesPyruvate kinase (PK) serves as a pivotal enzyme in the glycolytic pathway, facilitating the conversion of phosphoenolpyruvate (PEP) and adenosine diphosphate into pyruvate and ATP.In addition to its canonical role in glycolysis, PKM2 exhibits multifunctional capabilities as a protein kinase. In the nuclear milieu, PKM2 dimers function as active protein kinases, whereas the tetrameric form operates as an efficacious PK.In response to epidermal growth factor (EGF) stimuli, PKM2 directly phosphorylates and associates with histone H3. This interaction is requisite for K9 histone H3 acetylation, and such PKM2‐dependent chromatin modifications bear significant implications for brain carcinogenesis, tumor cell proliferation, cell cycle dynamics, and EGF‐mediated expression of cyclin D1 and c‐Myc.Furthermore, PKM2 modulates the G1/S phase transition by orchestrating the expression of cyclin D1 and effecting phosphorylation of the Bub3 constituent of the spindle assembly checkpoint complex (SAC). Intriguingly, Jiang et al. reported that glioblastoma prognosis correlates with the phosphorylation levels of Bub3 at the Y207 residue.The functionality of nuclear PKM2 extends beyond enzymatic catalysis to the regulation of gene expression, chromatin modification, and cell cycle progression, thereby implicating it in oncogenesis. Nevertheless, comprehensive investigations remain imperative for elucidating the intricate roles of PKM2 as a protein kinase.Emerging research posits that ROS exert a direct modulatory impact on PKM2, subsequently altering tumor metabolic dynamics. Under conditions of oxidative stress, the acetylation state of PKM2 diminishes, thereby resisting lysosomal degradation and contributing to pharmacological resistance in renal cell carcinoma.Phytochemicals such as Shikonin and alkannin, derived from traditional Chinese herbal medicine, have been identified as specific PKM2 inhibitors.+LDH is a pivotal enzyme that mediates the interconversion between pyruvate and lactate, while concurrently catalyzing the redox reaction involving NADH and NADTherapeutically, Galloflavin has been identified as a putative LDH inhibitor that induces cancer cell apoptosis by obstructing glycolysis and ATP synthesis.Within the TCA (tricarboxylic acid) cycle, fumarase, also known as FH, serves as an enzymatic catalyst facilitating the reversible hydration and dehydration of fumarate to malate, thereby promoting energy production via the generation of reduced NAD (NADH) Figures  and 2. RFBP acts as a rate‐limiting enzyme, orchestrating the hydrolysis of fructose 1,6‐bisphosphate to fructose 6‐phosphate, a pivotal step in gluconeogenesis — the biochemical antithesis of glycolysis. Vertebrates express two isozymic forms of FBP: FBP1, predominantly expressed in hepatic and renal tissues, and FBP2, which is primarily localized in skeletal muscle and mesenchymal tissues.3Since the seminal discovery of metabolic aberrations in neoplastic tissues, the field of cancer metabolism has witnessed significant advancements over the past several decades. Emerging therapeutic paradigms grounded in metabolic regulation have been developed and integrated into the clinical management of malignancies. These include the employment of small‐molecule inhibitors targeting pivotal metabolic pathways, the combination of metabolic interventions with immune checkpoint inhibitors, and multimodality treatment strategies that incorporate metabolic drugs into conventional oncological regimens such as surgical resection, radiation, and chemotherapy.3.1In the clinical armamentarium against cancer, metabolic chemotherapy agents are predominantly classified into two categories: folate antagonists and nucleotide base analogs. These compounds primarily exert their antineoplastic effects by obstructing nucleotide synthesis and thereby impeding cellular replication.3.2As elucidated in preceding discussions, key metabolic pathways such as the TCA cycle, one‐carbon metabolism, and glucose metabolism are instrumental in regulating nuclear processes including RNA modifications, DNA methylation, and histone modifications. These pathways supply essential metabolites that function as cofactors in these nuclear modifications. Additionally, the intermediates generated from these metabolic cycles contribute to nucleotide synthesis, ribose generation, and the production of purine and pyrimidine nucleotides.3.3In addition to metabolites that wield significant epigenetic influence on oncogenesis, metabolic enzymes serve as pivotal regulatory entities in the trajectory of tumor development. Evidence suggests that these enzymes undergo nuclear translocation, where they manifest kinase or nonkinase functions that modulate gene transcription, DNA damage repair, and posttranslational modifications, thereby influencing oncogene expression Table . Althoug3.4The advancement of scientific research has laid bare a myriad of nuclear protein, DNA, and RNA modifications that serve as cardinal regulators of nuclear function and the transmission of epigenetic information. HDAC inhibitors, including compounds such as Vorinostat (Zolinza), Belinostat, Panobinostat (Faridak), and Romidepsin (Istodax), have received approval from the United States Food and Drug Administration (US FDA) and have demonstrated clinical efficacy in oncological applications3.5In contrast to the more mature understanding, we possess concerning protein and DNA modifications, the scientific comprehension of RNA modifications remains in its formative stage. This nascent understanding has led to ongoing research endeavors that have yet to fully elucidate the complexities inherent in RNA modifications. As a result, pharmacological inhibitors targeting these modifications are still in their incipient phases and have generally not progressed to the stage of clinical trials Table . Neverth3.6Beyond the aforementioned therapeutic strategies, alternative approaches are under exploration in both preclinical and clinical settings. These include: (1) manipulating transcription factor regulation as a means of controlling or responding to metabolic aberrations, such as the role played by Sterol Regulatory Element‐Binding Proteins (SREBPs) in lipid metabolism, and transcription factors like p53 and c‐Myc.4Cellular metabolism and genomic regulation are inextricably intertwined, operating in a highly coordinated manner to facilitate rapid cellular adaptation to fluctuating extracellular nutrient conditions Figure . A failu+, serve as requisite precursors for these modifications while simultaneously affecting the activity of the enzymes that govern them.As elucidated in this review, a salient feedback loop involving metabolism, epigenetics, and gene regulation serves as a linchpin in the progression of malignancies Figure . Within Secondly, nuclear metabolic enzymes, exemplified by but not limited to PKM2, orchestrate gene transcription via distinct pathways, extending beyond their conventional metabolic roles. For instance, PKM2 is capable of phosphorylating histone H3 to directly modify chromatin architecture. Beyond these traditionally understood metabolic functions, metabolic enzymes can engage in direct interactions with epigenetic regulatory proteins  targeting nuclear metabolic enzymes; (2) modulating nuclear‐specific modifications; (3) interfering with the trafficking of metabolites or metabolic enzymes; and (4) targeting modulators of these metabolic enzymes. However, the ultimate efficacy of these novel therapeutic modalities is contingent upon an enhanced understanding of the specific metabolic requirements of tumor cells and the concomitant development of innovative antimetabolic strategies.The metabolic underpinnings of cancer are likely more expansive than currently anticipated, potentially influencing every facet of neoplastic progression. Advancements in research focusing on nuclear metabolic signaling—still in its nascent stage—will undoubtedly yield groundbreaking insights and perhaps paradigm shifts in our understanding of tumorigenesis. The identification of novel metabolic enzymes within the nuclear compartment, accompanied by a comprehensive elucidation of their functional roles and regulatory mechanisms, will enrich our understanding of tumor metabolism and highlight hitherto undiscovered targets for future pharmacological interventions.P. Y. and C. C. conceived the manuscript; Y. C., J. X., and X. L. wrote the initial draft; L. G., J. X., C. C., and Y. C. prepared the figure; C. C. revised the manuscript. All authors have read and approved the final manuscript.The authors declare no conflict of interest.Not applicable.