No description available.

No description available.

Cardiovascular disease (CVD) is the leading cause of death worldwide, and is projected to remain so for at least the next decade. Ever since its discovery in the urine and blood of children with inborn errors of metabolism, homocysteine (Hcy) at elevated plasma concentrations has been associated with CVD clinically and epidemiologically. Observational studies and meta-analyses have noted that changes in plasma Hcy by 5μM increase the odds ratio of developing coronary artery disease by 1.6-1.8 among other CVD. Clinical trials aimed at reducing plasma Hcy for benefit against development of subsequent cardiovascular events have had unconvincing results, but have moreover failed to address the mechanisms by which Hcy contributes to CVD. Recommendations from national agencies like the American Heart Association and the United States Preventive Services Task Force emphasize primordial prevention as a way to combat CVD. Reducing plasma Hcy as secondary and primary interventions does not fulfill this recommendation. In order to best understand the role of Hcy in CVD, an investigation into its mechanisms of action must be undertaken before measures of primordial prevention can be devised. Numerous experimental studies in the literature identify vascular endothelium as a target for the pathological effects of Hcy. Endothelial injury and impairment are contributory processes to atherosclerosis, and Hcy has been demonstrated to inhibit endothelial cell (EC) growth and proliferation through mechanisms involving cell cycle arrest, oxidative stress, and programmed cell death in vitro. Animal models have also confirmed that high levels of Hcy accelerate atherosclerotic plaque development and lead to impairment of vascular reendothelialization following injury. Hcy has been shown to have the opposite effect in vascular smooth muscle cells (SMC), causing their proliferation and again contributing to atherosclerosis. The cell-type specificity of Hcy remains to be understood, and among the aims of this research was to further characterize the effects of Hcy in EC. The overarching goal was discovery in order to direct future investigations of Hcy-mediated pathology. To begin, the first investigation considered the transcriptional and regulatory milieu in EC following exposure to Hcy. High-throughput screening using microarrays determined the effect of Hcy on 26,890 mRNA and 1,801 miRNA. Two different in vitro models of hyperhomocysteinemia (HHcy) were considered in this analysis. The first used a high dose of 500µ Hcy to mimic plasma concentrations of patients wherein the transsulfuration pathway of Hcy metabolism is impaired as in inborn cystathionine-ß-synthase deficiency. The other set of conditions used 50µ Hcy in the presence of adenosine to approximate impairment of the remethylation pathway of Hcy metabolism wherein s-adenosylhomocysteine accumulates, thus inhibiting s-adenosylmethionine formation and methylation reactions. These distinctions are important because most clinical trials do not distinguish between causes of HHcy, thereby ignoring the specific derangements underlying HHcy. mRNA and miRNA expression changes for both sets of treatment conditions identified CVD as a common network of Hcy-mediated pathology in EC. Moreover, methylation-specific conditions identified cell cycle modulation as a major contributory mechanism for this pathology, which agrees with recent findings in the literature. Analysis of significant mRNA changes and significant miRNA changes independently identified roles for Hcy in CVD and cell cycle regulation, thereby suggesting that miRNA may mediate the effects of Hcy in addition to gene expression changes alone. To investigate the role of Hcy in the cell cycle further, the next set of investigations considered the effect of Hcy under conditions approximating impaired remethylation in early cell cycle events. Previous studies have demonstrated that Hcy inhibits cyclin A transcription in EC via demethylation of its promoter. Conversely, Hcy induces cyclin A expression in SMC, again making the case for a cell type-specific mechanism in EC. Preceding cyclin A transcription and activation, canonical events in the early cell cycle include D-type cyclin activation, retinoblastoma protein (pRB) phosphorylation, and transcription factor E2F1 activation. In a series of in vitro experiments on EC, it was seen that Hcy inhibits expression of cyclin D2 and cyclin D3, but not cyclin D1. Next, pRB phosphorylation was seen to be decreased following treatment with Hcy. This also led to decreased E2F1 expression. However, this series of events could be reversed with E2F1 supplementation, allowing the cell cycle to proceed. As Hcy exerts a number of its effects via regulation of gene transcription, a final series of investigations aimed to predict potential targets of Hcy by examining patterns of transcription factor binding among known targets of Hcy regulation. Gene promoters of Hcy-modulated genes were analyzed in order to determine common transcription factors that potentially control their regulation. The locations of CpG-rich regions in promoters were identified to determine which regions would be most susceptible to regulation by DNA methylation. Next, high-throughput next-generation sequencing (NGS) and bisulfite NGS was performed for DNA from EC treated with Hcy in order to determine methylation changes after Hcy treatment. A number of potential transcription factors and their binding sites were identified as potential mediators of Hcy-mediated gene regulation. Taken together, these investigations represent an exploration of Hcy-mediated pathology in CVD, by focusing upon novel regulatory mechanisms in EC. Objective high-throughput arrays identified roles for Hcy in CVD and cell cycle pathways regulated by miRNA and gene expression, which were confirmed experimentally in vitro. These observations led to an investigation and identification of common transcription factors that potentially regulate Hcy-altered gene expression. This framework may be used to guide future investigations into the complex pathological network mediating the effects of Hcy in CVD. First, identification of a role for miRNA in mediating the effects of Hcy represents a novel regulatory mechanism, heretofore largely unexplored. Next, expanding the role of Hcy in EC cell cycle regulation to identify upstream mediators greatly adds to the published literature. Finally, noting that these changes center upon transcriptional and post-transcriptional regulation gives import to developing methods to characterize promoter and transcription factor regulation. The investigations presented herein and their results provide evidence that the future of Hcy research is vibrant, relevant, and not nearly surfeit. / Pharmacology

Cyclin-Dependent Kinase 9 (CDK9) as part of the PTEFb complex promotes transcriptional elongation by promoting RNAPII pause release. We now report that, paradoxically, CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell screen, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression and cell differentiation, along with activation of endogenous retrovirus (ERV) genes. CDK9 inhibition results in dephorphorylation of the SWI/SNF protein SMARCA4 and represses HP1α expression, both of which contribute to gene reactivation. Based on gene activation, we developed the highly selective and potent CDK9 inhibitor MC180295 (IC50 =5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes with the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer. / Molecular Biology and Genetics

Yes / Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo- Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics.

No / We organized the first International Symposium on Skin Epigenetics at the Centre for Skin Sciences at the University of Bradford (West Yorkshire, UK) on 2nd and 3rd April 2012. The goal of the Symposium was to bring together two research communities—skin and chromatin biologists—and discuss the most important aspects of epigenetic regulatory mechanisms that control skin development and regeneration. The symposium was attended by more than 80 participants from countries across Europe, Australia, Japan, Singapore, and USA, and representing academic institutions and industry. Epigenetic regulation of gene expression programs in the skin is a novel trend in research in cutaneous biology, and several landmark papers arising in the field were published recently (reviewed in Botchkarev et al., 2012; Botchkareva, 2012; Frye and Benitah, 2012; Yi and Fuchs, 2012; Zhang et al., 2012). The Symposium program included six Keynote lectures, the inaugural John M. Wood Memorial Lecture, and six sessions that covered major levels of epigenetic regulation.

No / The nucleus is a complex and highly compartmentalized organelle, which undergoes major organization changes during cell differentiation, allowing cells to become specialized and fulfill their functions. During terminal differentiation of the epidermal keratinocytes, the nucleus undergoes a programmed transformation from active status, associated with execution of the genetic programs of cornification and epidermal barrier formation, to a fully inactive condition and becomes a part of the keratinized cells of the cornified layer. Tremendous progress achieved within the past two decades in understanding the biology of the nucleus and epigenetic mechanisms controlling gene expression allowed defining several levels in the regulation of cell differentiation–associated gene expression programs, including an accessibility of the gene regulatory regions to DNA–protein interactions, covalent DNA and histone modifications, and ATP-dependent chromatin remodeling, as well as higher-order chromatin remodeling and nuclear compartmentalization of the genes and transcription machinery. Here, we integrate our current knowledge of the mechanisms controlling gene expression during terminal keratinocyte differentiation with distinct levels of chromatin organization and remodeling. We also propose directions to further explore the role of epigenetic mechanisms and their interactions with other regulatory systems in the control of keratinocyte differentiation in normal and diseased skin.

Στην παρούσα διδακτορική διατριβή δείχθηκε ότι το γονίδιο της ανθρώπινης καλλικρεΐνης 6 μπορεί να ενεργοποιηθεί φαρμακολογικά σε καρκινικές κυτταρικές σειρές μαστού και μελετήθηκε ο μοριακός μηχανισμός της ενεργοποίησης. Το cDNA της ανθρώπινης καλλικρεΐνης 6 αρχικά κλωνοποιήθηκε με την τεχνική της διαφορικής παράθεσης των mRNAs βάσει της υπερέκφρασης σε πρωτοπαθή όγκο μαστού σε σχέση με την πλήρη αποσιώπηση στην μετάστασή του στον πνεύμονα, καθώς και στις περισσότερες μεταστατικές καρκινικές σειρές μαστού και δείγματα ιστών. Λόγω της καταστολής της έκφρασής της σε μεταστατικούς όγκους του μαστού, θεωρήθηκε ότι η κωδικοποιούμενη πρωτεΐνη (hK6)-μια νέα σερινοπρωτεάση- θα μπορούσε να έχει ογκοκατασταλτική δράση. Πρόσφατα δεδομένα δείχθουν ότι η hK6 αντιπροσωπεύει έναν νέο μοριακό δείκτη του καρκίνου, αφού αυξημένες συγκεντρώσεις της στον ορό είναι διαγνωστικές για τον καρκίνο των ωοθηκών και μπορούν να χρησιμοποιηθούν για την παρακολούθηση της θεραπευτικής αγωγής. Οι φυσιολογικές λειτουργίες της hK6 δεν έχουν βρεθεί. Πρόσφατα, αλλαγές στην έκφραση του γονιδίου KLK6 έχουν συσχετιστεί με την παθογένεση των πιο κοινών νευροεκφυλιστικών νόσων, όπως Alzheimer’s και Parkinson’s. Σημαντικό, είναι το γεγονός της αποικοδόμησης της μυελίνης από την hK6, που πιστοποιεί την συμμετοχή της στην σκλήρυνση κατά πλάκας. Για τους παραπάνω λόγους η hK6, καθώς και το γονίδιο KLK6 αποτελούν ένα σημαντικό θεραπευτικό στόχο. Στην παρούσα εργασία, δείχθηκε ότι οι μηχανισμοί που ρυθμίζουν την μεταγραφή καθώς και την ιστο-εξειδικευμένη έκφραση του γονιδίου KLK6 περιλαμβάνουν την δραστικότητα δύο υποκινητών. Επίσης κλωνοποιήθηκαν νέες ισομορφές του γονιδίου που προκύπτουν από εναλλακτικό μάτισμα και αντιστοιχούν στο 10-20% των KLK6 mRNAs. Προηγούμενη έρευνα δεν είχε παρατηρήσει εκτεταμένες γονιδιωματικές αλλαγές σε καρκινικούς όγκους στην γενετικό τόπο 19q13.4 που εδρεύει το γονίδιο KLK6, και σε συνδυασμό με την πιθανή ογκοκατασταλτική δράση της hK6, διερευνήθηκε η συμμετοχή επιγενετικών μηχανισμών στην αποσιώπηση της έκφρασης του γονιδίου KLK6. Κατεργασία των καρκινικών κυτταρικών σειρών μαστού T47D και MDA-MB-231 με το απομεθυλιωτικό αντιδραστήριο 5-αζα-2΄-δεοξυκυτιδίνη επανενεργοποίησε την έκφραση του γονιδίου KLK6. Η τριχοστατίνη Α, αναστολέας των αποακετυλασών των ιστονών, επανενεργοποίησε την έκφραση KLK6 μόνο στα MDA-MB-231 κύτταρα. Επίσης, βρήκαμε ότι η καταστολή της έκφρασης KLK6 στις καρκινικές κυτταρικές σειρές μαστού συνδέεται με την μεθυλίωση συγκεκριμένων δινουκλεοτιδικών αλληλουχιών CpGs που βρίσκονται στον εγγύς υποκινητή του γονιδίου και σε θέσεις πρόσδεσης του μεταγραφικού παράγοντα Sp1. Η αποσιώπηση της έκφρασης λαμβάνει χώρα με την πρόσδεση του εξαρτώμενου από μεθυλίωση μεταγραφικού καταστολέα MeCP2 και την δημιουργία ετεροχρωματινικής δομής λόγω αποακετυλίωσης των ιστονών. Επειδή η επιγενετική αποσιώπηση του γονιδίου KLK6 υποδηλώνει ογκοκατασταλτικό ρόλο στον καρκίνο του μαστού, διαμολύνθηκε σταθερά το KLK6 cDNA στην μεταστική σειρά MDA-MB-231 με σκοπό τη διαπίστωση του πιθανού αυτού ρόλου. Τα σταθερά διαμολυσμένα κύτταρα που προέκυψαν, είχαν μικρότερο ρυθμό πολλαπλασιασμού, ενώ δεν μπορούσαν να σχηματίσουν αποικίες σε μαλακό άγαρ. Συμπερασματικά στην παρούσα διατριβή δείχθηκε ότι το γονίδιο KLK6 αποτελεί πιθανό ογκοκατασταλτικό γονίδιο, που αποσιωπάται σε καρκινικές σειρές μαστού μέσω μεθυλίωσης του γονιδιωματικού DNA, καθώς και δημιουργίας ετεροχρωματινικής δομής στον εγγύς υποκινητή του. Η φαρμακολογική ενεργοποίηση της έκφρασης του γονιδίου KLK6 μέσω επιγενετικών φαρμάκων, είναι πιθανό να ανοίξει νέους δρόμους για την αντιμετώπιση του καρκίνου του μαστού. / In the present thesis it was shown that the human kallikrein 6 gene is pharmacologically modulated in breast cancer cell lines and the molecular mechanism accounting for the modulation was analyzed. The cDNA encoding human kallikrein 6 (protease M) was originally cloned by mRNA differential display as being over expressed in a primary breast tumor but completely inactivated in its lung metastasis, and in the majority of metastatic breast cancer cell lines and tissue specimens. Based on this expression pattern, it was suggested that the encoded protein (hK6)-a novel serine protease-could play a suppressor role in cancer progression. Recent evidence suggests that hK6 represents a novel cancer biomarker, since elevated serum concentrations of hK6 are diagnostic of ovarian cancer and can be exploited for monitoring therapeutic response to treatment. The physiological function(s) of hK6 have not been elucidated. Recently, aberrant expression of the KLK6 gene has been implicated in the pathogenesis of most common neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. In addition, hK6 is involved in enhanced proteolysis of myelin basic protein associated with multiple sclerosis. Therefore, hK6 and KLK6 gene, represent potential therapeutic targets for pharmacological intervention. In the present study, we have shown that the mechanisms regulating KLK6 transcription and tissue-specific expression involve the action of two different promoters. Also, new KLK6 splice variants were cloned, and shown to account for 10-20% of all KLK6 mRNA species. Previous study had shown no gross genomic alterations in tumor speciments in the KLK6 genomic locus 19q13.4, and in accordance with putative tumor suppressor activity the involvement of epigenetic mechanisms in KLK6 gene silencing in breast cancer was studied. Treatment of KLK6-negative T47D and MDA-MB-231 human breast cancer cell lines with the demethylating agent 5-aza-2\\\\\\\\

Le neuroblastome pédiatrique à haut risque est incurable malgré l’intensification des traitements. Chez le patient, les cellules de neuroblastome échappent à l’activité anticancéreuse des cellules immunitaires Natural Killer (NK). Or, lorsque cultivées in vitro en monocouche (2D), les cellules de neuroblastomes redeviennent sensibles à l’activité cytotoxique des cellules NK ce qui ne reflètent pas leur résistance dans les tumeurs in situ. Nous faisons l'hypothèse que lorsque cultivées en 3D sous forme de sphéroïdes, les cellules de neuroblastome pourraient retrouver certaines caractéristiques qui les rendraient plus représentatives des tumeurs in situ au niveau immunologique. Ainsi, un tel modèle préclinique pourrait mieux refléter la résistance aux cellules NK et servir de modèle de criblage pour la découverte de médicaments potentialisant la cytotoxicité des cellules NK. Pour répondre à cette question, nous avons développé un système de culture cellulaire 3D utilisant plusieurs lignées cellulaires de neuroblastome. À ce système, une co-culture en 3D avec une lignée de cellules Natural Killer (NK92) a été mise en place. Nous avons mis en évidence que les sphéroïdes de neuroblastome présentent des changements d’expression de certains gènes qui sont retrouvées chez les patients ainsi qu’une plus grande résistance à l’activité cytotoxique des cellules NK92 en comparaison avec les lignées en monocouche. Les co cultures de sphéroïdes ont été exposées à des inhibiteurs de protéines impliquées à différents niveaux de l’épigénome afin de découvrir des médicaments qui sensibiliseraient les cellules de neuroblastome à l’activité cytotoxique des NK92. Une différence dans la sensibilité aux médicaments entre les sphéroïdes et les cellules en 2D ainsi qu’en monoculture ou en co-culture a été observée et certains composés ont été identifiés en vue de potentialiser l’activité des cellules NK92. Ainsi, nos études ont permis de mieux comprendre les mécanismes impliqués dans la résistance des cellules du neuroblastome à l’activité cytotoxique des cellules NK dans un modèle plus représentatif de la tumeur in situ. / High-risk pediatric neuroblastoma remains incurable despite intensified treatments. In patients, neuroblastoma cells evade the anti-cancer activity of Natural Killer (NK) immune cells. However, when cultured in vitro in a monolayer (2D), neuroblastoma cells become sensitive to the cytotoxic activity of NK cells, which does not reflect their resistance in tumors in situ. We hypothesize that when cultured in 3D in the form of spheroids, neuroblastoma cells could regain certain characteristics that would make them representative of tumors in situ at the immunological level. Thus, such a preclinical model could better reflect NK cell resistance and serve as a screening model for drug discovery to discover a treatment that can potentiate NK cell cytotoxicity. To answer this question, we developed a 3D cell culture system using several neuroblastoma cell lines. To this system, a 3D coculture model with a Natural Killer (NK92) cell line was set up. We have shown that neuroblastoma spheroids develop changes in the expression of certain genes that are found in patients as well as greater resistance to NK92 cells compared to monolayer cell lines. Spheroid cocultures were exposed to inhibitors of proteins involved at different levels of the epigenome to discover drugs that would sensitize neuroblastoma cells to the cytotoxic activity of NK92. A difference in drug sensitivity between spheroids and cells in 2D as well as in monoculture or coculture was observed and some compounds were identified to potentiate the activity of NK92 cells. Thus, our studies have provided a better understanding of the mechanisms involved in the resistance of neuroblastoma cells to the cytotoxic activity of NK cells in a more representative model of the tumor in situ.

Résumé : La néphropathie diabétique (ND) est une complication microvasculaire du diabète évoluant ultimement en insuffisance rénale et l’hyperglycémie est connue comme étant l’un des facteurs de risques. De larges études cliniques, tel que le DCCT et l’UKPDS, ont montré que si le contrôle intensif de la glycémie se faisait de façon précoce, il serait possible de retarder le développement de la ND. Cependant, les résultats de l'EDIC montrent que si ce contrôle intensif se faisait plus tardivement, suite à une période d’hyperglycémie, il n’empêcherait plus sa progression. Les podocytes ont un rôle critique dans le maintien des fonctions rénales et leur apoptose corrèle de façon très spécifique avec la progression de la ND. Récemment, nous avons rapporté que SHP-1, une protéine tyrosine phosphatase, était augmentée en concentrations élevées de glucose (HG), menant à une inhibition des voies de signalisation de l'insuline. Notre hypothèse est que l’augmentation de l’expression de SHP-1 causée par l’hyperglycémie persiste même après réduction des niveaux de glucose, phénomène de mémoire hyperglycémique, causant une résistance à l'insuline, la mort des podocytes et une absence de réversibilité liée à la progression de ND. Les résultats in vivo montrent que la fonction et la pathologie rénale continuent de progresser et ce en dépit de la normalisation des niveaux de glucose avec implants d’insuline de 5 à 7 mois d’âge La progression de la pathologie corrèle avec le maintien de l’augmentation de l’expression de SHP-1, contribuant au maintien de l’inhibition des voies de l’insuline. En culture, des podocytes murins exposés en HG pendant 96 h et ensuite exposés en condition normale de glucose(NG) pour les dernières 24 h montrent une persistance de l’inhibition des voies de signalisation de l’insuline qui corrèle avec l’augmentation persistante de l’expression et l’activité phosphatase de SHP-1. L’activité des caspases 3/7 dans les podocytes est plus élevée lorsque ceux-ci sont exposés en HG qu’en NG. Le retour en NG pour les dernières 24 h n’a aucun effet bénéfique à réduire l’activité des caspases 3/7. Finalement, l’analyse épigénétique a été suggérée comme étant une explication du phénomène de mémoire hyperglycémique. La monométhylation de la lysine 4 de l’histone 3 (H3K4me1), un marqueur d’activation génique, est augmentée sur le promoteur de SHP-1 en HG et demeure élevée malgré le retour en NG pendant les dernières 24 h. En conclusion, l’hyperglycémie engendre une augmentation persistante de SHP-1 due possiblement à des modifications épigénétiques, causant le maintien de l’inhibition les voies de signalisation de l’insuline même après un retour à des niveaux normaux de glucose, contribuant à la progression de la ND. / Abstract : Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Renal podocytes apoptosis induced by hyperglycemia is an early event of DN. Clinical studies have shown that intensive blood glucose control reduced the development of DN but is not sufficient, if started late, to prevent its progression, introducing the concept of “hyperglycemic memory”. We have recently published that the tyrosine phosphatase SHP-1 is elevated in renal cortex of type 1 diabetic mice (Akita), contributing to insulin unresponsiveness and DN. We hypothesized that SHP-1 expression remains elevated regardless of systemic blood glucose normalization, and is responsible for hyperglycemic memory in podocytes leading to DN progression. In vivo contribution of SHP-1 in hyperglycemic memory was evaluated using Akita mice treated with insulin implants after 4 months of diabetes. Both urinary albuminuria and glomerular filtration rate were significantly increased in diabetic mice compared to non-diabetic mice and remained elevated despite normalization of blood glucose levels. Renal dysfunction was associated with a persistent increase of SHP-1 expression in renal cortex and inhibition of insulin action that were not normalized following insulin implants. Mouse podocytes were cultured in normal (5.6mM; NG), high glucose concentrations (25mM; HG) for 120 h or HG (96 h) followed by NG for an additional 24 h (HG+NG). We observed that Akt and ERK phosphorylation induced by insulin was inhibited in HG and were not restored despite returning glucose level to 5.6 mM after the HG period. This inhibition was associated with persistent increase of SHP-1 expression and phosphatase activity, leading to insulin signaling pathway inhibition. Moreover, caspase 3/7 activity in podocytes exposed to HG was higher than in podocytes cultured in NG and returning glucose concentrations to normal range for the last 24 h after the 96 h HG exposure had no effect on reducing caspase 3/7 activity. Epigenetic changes were studied to explain the hyperglycemic memory effect. On SHP-1 promoter, H3K4me1 levels, an activation mark, tended to be more elevated in podocytes exposed to HG and were maintained despite returning to NG levels after the HG conditions. In conclusion, hyperglycemia induces persistent and epigenetic changes of SHP-1 causing insulin unresponsiveness in the podocytes contributing to DN progression.