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Additional file 1: Figure S1. Identification of proximal interactors of DOT1L via BioID and their effect on reprogramming. (a) Replicate immunoblot of Fig. 1b. (b) CRAPome analysis of WT-DOT1L proximal proteins identified via BioID assay. Y-axis shows the PSM values from MS data. (c) mRNA levels of shRNA targeted genes were assessed via qRT-PCR. β-actin was used as an internal control and gene expression levels are normalized to control shFF (firefly luciferase targeting shRNA) expressing cells. (d) mRNA levels of shAF9 targeted genes were assessed via qRT-PCR. β-actin was used as an internal control and gene expression levels are normalized to control shFF (firefly luciferase targeting shRNA) expressing cells. (e) Fold change in the number of Tra-1-60 positive colonies upon shAF9 expression. P values were determined by one sample t-test; * P

Embriyonik kök hücreler (EKH) vücutta bulunan bütün somatik hücreleri oluşturabilmekapasitesine sahiptir. OCT4, SOX2, KLF4 ve cMYC (OSKM) transkripsiyonfaktörlerinin farklılaşmış hücrelere sunulması, yeniden programlamayı uyarıp EKHbenzeriuyarılmış plüripotent kök (uPKH) hücrelerin oluşmasını sağlamaktadır.Yeniden programlama, birden çok aşamalı bir süreçtir ve her aşamasında belirlimoleküler değişikliklerin gerçekleşmesi gerekmektedir. Bununla birlikte, kromatindüzenleyicilerinin, somatik hücrelerin gen ifadeleri üzerindeki etkileri ve yenidenprogramlamayı nasıl etkilediği konusunda birçok cevaplanmamış soru bulunmaktadır.SETD2, aktif transkripsiyon ile ilişkilendirilen Histone H3 lizin 36 (H3K36) kalıntısınıntri-metilasyonundan sorumlu olduğu bilinen tek kromatin düzenleyicidir. H3K36metilasyonunun yeniden programlama üzerindeki etkisi halen bilinmemektedir. Busebeple SETD2'nin insan fibroblast hücrelerinin yeniden programlanması üzerindekirolünü, shRNA-tabanlı gen susturulması ya da gRNA-tabanlı gen çıkarılmasıstratejileri ile inceledik. Buna ek olarak, H3K36 metilasyonu kaybının gen ifadesi ağınınasıl etkilediğini anlamak amacı ile, OSKM-sunumu öncesi ve sonrası elde edilenSETD2 ifadesi azaltılmış hücrelere RNA dizileme analizi uyguladık.Elde ettiğimiz sonuçlara göre, SETD2 ifadesin baskılanması, genel H3K36me3ifadesinde azalmaya yol açmakta ve bu soytür, fibroblast hücrelerinin OSKM-tabanlıyeniden programlaması arttırmaktadır. Ek olarak, SETD2 ifadesindeki baskılanma,OS-tabanlı yeniden programlama sürecinde KLF4 ve cMYC yerine geçmek içinyeterlidir. SETD2 ifadesindeki azalış, yeniden programlamanın erken evrelerindeetkili iken, RNA dizileme ile de anlaşıldığı üzere, bu durum somatik gen ifadelerininazalmasına yol açmaktadır. Bu soytür, H3K36 metilasyonunu baskın olarakengelleyen histone H3 mutantının aşırı ifadesi sonucunda da görülmektedir. Busebeple, SETD2'nin baskılanması sonucunda görülen H3K36me3 aktif işaretindekiazalışın, OSKM faktörlerinin fibroblast-özgü ifadeler üzerindeki baskılayıcı etkisinekatkıda bulunduğunu öne sürmekteyiz.Elde ettiğimiz sonuçlar göz önüne alındığında, SETD2'nin yeniden programlama içinbir engel olduğu ve ifadesindeki azalışın somatik gen ifadelerini baskılaması yolu ileyeniden programlamayı olumlu etkilediği görülmektedir. Embryonic stem cells (ESCs) have the capacity to form all types of somatic cells inthe body. Introduction of four transcription factors OCT4, SOX2, KLF4 and cMYC(OSKM) into somatic cells induces reprogramming to generate ESC-like cells.Reprogramming is a step-wise process which requires several molecular changes.One of these is the remodeling of global chromatin landscape of somatic cells to gainpluripotency. However, there remain many unanswered questions regarding theinfluence of chromatin regulators on the gene expression network of somatic cells andhow they affect reprogramming.SETD2 is the only chromatin modifier responsible for generating the H3 Lysine 36(H3K36) trimethylation, a histone mark that is associated with active transcription. Therole of H3K36 methylation in reprogramming remains unknown. Therefore, weinvestigated the role of SETD2 in reprogramming via shRNA-mediated knock-downor gRNA-mediated knock-out strategies in human fibroblasts. We also performedRNA sequencing on SETD2-depleted cells before and after OSKM-induction tounderstand how loss of H3K36 methylation affects gene expression networks.Our results demonstrate that, global H3K36me3 decreases upon SETD2 depletionand this phenotype enhances OSKM-mediated reprogramming of fibroblasts.Additionally, SETD2 depletion is sufficient to replace KLF4 and cMYC in OS-mediatedreprogramming. Downregulation of SETD2 is effective in the early stages ofreprogramming and leads to a decrease in somatic specific gene expression levels.This phenotype is also observed upon overexpression of a histone H3 mutant thatdominantly blocks H3K36 methylation. We suggest that reduction of H3K36me3active marks upon SETD2 depletion contributes to OSKM-mediated suppression offibroblast specific genes.Taken together these results indicate that SETD2 is a roadblock for reprogrammingand reduction of its expression has a positive effect on reprogramming throughsuppression of somatic specific genes. 208

Nöroblastom (NB) sempatik sinir sistemine farklılaşan nöral krest hücrelerinden köken alan embriyonik bir tümördür. Spontan remisyondan hızlı metastatik progresyona kadar varan farklı klinik davranışlar gösterir. NB, karyotipik açıdan segmental kromozom aberasyonları ile karakterizedir. Sık gözlenen reküran kromozom anomalileri 1 no?lu kromozomun kısa kolunda (1p) ve 11 no?lu kromozomun uzun kolunda (11q) gözlenen kayıplar; 17 no?lu kromozomun uzun kolundaki (17q) kazanımlardır. NB olgularının % 22?sinde MYCN geni amplifikasyonu ileri hastalık evresi, metastatik hastalık ve agresif klinik seyirle birliktelik göstermektedir. Ayrıca, 1p, 11q delesyonları ve 17q duplikasyonu kötü prognozla ilişkilendirilmiştir. Buna karşın, söz konusu kromozom segmentlerinde, NB patogenezinde rol oynayan gen ya da genler bugüne kadar tanımlanamamıştır. Erişkin tümörlerinde gözlenen kanser genleri de NB tümörlerinde tutulum göstermemektedir. Bu çalışmanın çıkış noktası NB?deki bu prognostik faktörlerin sitogenetik anomaliler haricinde başka etmenlere dayandırılabileceği düşüncesidir. Günümüzde kanser üzerine yapılan araştırmalarda karsinogenez sürecinde epigenetik faktörlerin de rol oynadığının bulunması ve NB tümörlerinde bu tarz çalışmaların fazla olmaması, NB tümör fenotipindeki bu çeşitliliğe epigenetik faktörlerin neden olabileceği sorusunu doğurmuştur. DNA metilasyonu normal gelişim sürecinde rol oynayan bir epigenetik faktördür ve metilasyon örüntünün bozulması karsinogenezde doğrudan rol oynamaktadır. Buna ek olarak histon modifikasyonlarının, protein komplekslerinin DNA?ya bağlanma sürecini olumlu ve olumsuz yönde etkiledikleri, bu sebeple genom üzerinde belirli bölgelerin gen ifadesinde arttırıcı veya susturucu etkisi olduğu bilinmektedir.Proje kapsamında epigenetik değişikliklere yol açan 84 tane genin ifade değişimleri gerçek zamanlı polimeraz zincir reaksiyonu analizleri ile belirlenmiştir. Analiz sırasında MYCN amplifikasyonu görülmeyen iki (2) adet NB hücre hattı kontrol grubu olarak kullanılmış ve MYCN amplifikasyonu gözlenen beş (5) adet NB hücre hattı üzerinde ilgili genlerin ifade değişimleri saptanmıştır.??Ct formülü kullanılarak yapılan hesaplama sonucunda, hedef grup ile kontrol grubu karşılaştırıldığında AURKB geninin ifadesinde 6.45 kat, ESCO2 geninin ifadesinde 6.75 kat, HDAC2 geninin ifadesinde 5.41 kat, PRMT8 geninin ifadesinde 5.80 kat ve RNF2 geninin ifadesinde 4.12 kat artış gözlenmiştir. Aynı şekilde yapılan karşılaştırma sonucunda HDAC9 geninin ifadesinde 4.51 ve NEK6 geninin ifadesinde ise 4.66 kat azalış gözlenmiştir. Neuroblastoma(NB) is an embryonic tumor originated from neural crest cells which are origined to sympathetic nervous system. The clinic feature of this tumor has a wide spectrum changing from spontaneous remission to metastatic progression. NB is characterized by segmental chromosome abnormalities. The most common recurrent chromosome abnormalities in NB tumors are the deletion of the short arm of the chromosome 1(1p), deletion of the long arm of the chromosome 11(11q) and addition of the long arm of the chromosome 17(17q). In the 22% of the NB cases, MYCN amplification is associated with advanced disease stage, metastasis and aggressive clinical behavior. Moreover, the deletions of 1p and 11q and the duplication of 17q are associated with poor prognosis. Nevertheless, there has not been any genes are detected in these chromosome segments that might play a role in NB pathogenesis. Also the cancer genes characterized in adult tumors are not involved in NB tumors.The origin of this study is that, the prognostic factors of NB may arise from different factors rather than cytogenetic abnormalities. Nowadays, it is known that the epigenetic factors play role in cancer progression and there has been a limited study for the effect of the epigenetic mechanisms on NB development. This information led to the question that the diversity of NB tumor phenotypes can be caused by epigenetic factors. DNA methylation is an epigenetic factor that plays a role in normal development process and the corruption of methylation patterns plays a direct role in carcinogenesis. In addition to this, it is known that the histone modifications regulate the binding of the protein complexes to DNA in a positive or negative manner, so that specific sites on the genome is said to have enhancing or silencing effects the gene expression.In this project, the expression profiles of 84 genes involved in the epigenetic mechanism are detected by real time polymerase chain reaction analysis. Two (2) MYCN-non-amplified cell lines were selected as control group and five (5) MYCN-amplified cell lines were analyzed in order to determine the gene expression profiles.The relative gene expressions were calculated by ??Ct formula and the results gained from the comparison of MYCN-non-amplified and MYCN-amplified cell lines demonstrated that there has been a 6.45-fold increase in AURKB, 6.75-fold increase in ESCO2, 5.41-fold increase in HDAC2, 5.80-fold increase in PRMT8, 4.12-fold increase in RNF2, 4.51-fold decrease in HDAC9 and 4.66-fold decrease NEK6 respectively. 123

Background: the histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. Results: we employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. Conclusions: our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells., Scientific and Technological Research Council of Turkey (TÜBİTAK) Project; European Union (EU); Horizon 2020; Seventh Framework Programme (FP7/2007-2013) under REA Grant Agreement; People Programme (Marie Curie Actions); Arthritis Research UK; EMBO Installation Grant; Newton Advanced Fellowship; Cancer Research UK; Leducq Foundation LEAN Project

Friedreich's ataxia (FRDA) is a rare neurodegenerative disorder which is caused by triplet repeat expansion (GAA) in the first intron of FXN gene. In this present study, we generated induced pluripotent stem cells (iPSC) lines from fibroblasts of three unrelated FRDA patients using integration-free episomal vectors. All iPSC lines express the pluripotency markers such as OCT4 and SSEA4, display normal karyotypes and can differentiate into all three germ layers via in vivo teratoma formation assay., NA

Complete hydatidiform mole (HM) is a gestational trophoblastic disease resulting in hyperproliferation of trophoblast cells and absence of embryo development. Mutations in the maternal-effect gene NLRP7 are the major cause of familial recurrent complete HM. Here, we established an in vitro model of HM using patient-specific induced pluripotent stem cells (iPSCs) derived trophoblasts harboring NLRP7 mutations. Using whole transcriptome profiling during trophoblast differentiation, we showed that impaired NLRP7 expression results in precocious downregulation of pluripotency factors, activation of trophoblast lineage markers, and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. Interestingly, we found that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition corrected the excessive trophoblast differentiation of patient-derived iPSCs. Our human iPSC model of a genetic placental disease recapitulates aspects of trophoblast biology, highlights the broad utility of iPSC-derived trophoblasts for modeling human placental diseases and identifies NLRP7 as an essential modulator of key developmental cell fate regulators., Scientific and Technological Research Council of Turkey (TÜBİTAK); Boğaziçi University Research Funding Office; EMBO Installation Grant

Summary Organoid technologies have become a powerful emerging tool to model liver diseases, for drug screening, and for personalized treatments. These applications are, however, limited in their capacity to generate functional hepatocytes in a reproducible and efficient manner. Here, we generated and characterized the hepatic organoid (eHEPO) culture system using human induced pluripotent stem cell (iPSC)-derived EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, we modeled citrullinemia type 1, a urea cycle disorder caused by mutations in the argininosuccinate synthetase (ASS1) enzyme. The disease-related ammonia accumulation phenotype in eHEPOs could be reversed by the overexpression of the wild-type ASS1 gene, which also indicated that this model is amenable to genetic manipulation. Thus, eHEPOs are excellent unlimited cell sources to generate functional hepatic organoids in a fast and efficient manner., Graphical Abstract, Highlights • iPSC-derived EpCAM+ endodermal cells generate functional hepatic organoids (eHEPOs) • eHEPOs can be stably maintained in long-term culture • An organoid-based model for a urea cycle disorder (citrullinemia) is generated • Genetic manipulation of disease-specific eHEPOs rescues disease phenotype, Erdal and colleagues generated a hepatic organoid (eHEPO) culture system using human iPSC-derived EpCAM-positive endodermal cells. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, a urea cycle disorder, citrullinemia, was modeled and disease phenotype was rescued via overexpression of wild-type ASS1 gene.