Epigenetics and chromosome segregation in human pre-implantation embryos

Abstract Chapter 1 Currently, the average pregnancy rate per embryo transfer after in vitro fertilization (IVF) is around 32%. In order to achieve better results in the future, we need to gain knowledge on all aspects of the treatment, including pre-implantation embryo development. In this thesis, we describe the research we performed into epigenetics and chromosome segregation in human pre-implantation embryos derived from IVF. The term ‘epigenetics’ refers to heritable marks on the genome, such as DNA methylation and histone modifications. These marks are essential for chromosome structure, chromosome segregation and gene expression. Chromosome segregation is the process in which duplicated chromosomes are equally separated over two cells during cell division. Chromosomal abnormalities are detected at high frequencies in human pre-implantation embryos. This suggests that mechanisms regulating chromosome segregation are less functional during the first cell divisions of an embryo. Next to that, epigenetic marks, which are also important for correct chromosome segregation, are different in oocytes and spermatozoa and need to be re-established in early embryos. The research described in this thesis aimed to investigate both the mechanisms regulation chromosome segregation and the re-establishmen

Synchronous and metachronous urothelial carcinoma of the upper urinary tract and the bladder: Are they clonally related? A systematic review

Purpose: Following radical nephroureterectomy for upper urinary tract urothelial carcinoma (UTUC), intravesical recurrence (IVR) is found in 22% to 47% of patients. Patients with a primary urothelial carcinoma of the bladder (UCB) have an increased risk of a future UTUC (1%–5%). Paired UTUC and UCB might represent clonally related tumors due to intraluminal seeding of tumor cells or might be separate entities of urothelial carcinoma caused by field cancerization. We systematically reviewed all the relevant literature to address the possible clonal relation of UTUC and paired UCB. Materials and Methods: MEDLINE, EMBASE, and COCHRANE databases were systematically searched for relevant citations published between January 2000 and July 2019. This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Of 5038 citations identified, 86 full papers were screened, and 9 studies met the inclusion criteria. Results: The populations studied and the molecular techniques used to assess clonality of UTUC and paired UCB differed largely over time. Eight studies reported on primary UTUC and meta- or synchronous IVR without a history of UCB. A total of 118 tumors

Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications

The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos

DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus

Background: DNA methylation is of pivotal importance during development. Previous genome-wide studies identified numerous differentially methylated regions upon differentiation of stem cells, many of them associated with transcriptional start sites. Results: We present the first genome-wide, single-base-resolution view into DNA methylation dynamics during differentiation of a mammalian epithelial stem cell: the mouse small intestinal Lgr5+ stem cell. Very little change was observed at transcriptional start sites and our data suggest that differentiation-related genes are already primed for expression in the stem cell. Genome-wide, only 50 differentially methylated regions were identified. Almost all of these loci represent enhancers driving gene expression in the differentiated part of the small intestine. Finally, we show that binding of the transcription factor Tcf4 correlates with hypo-methylation and demonstrate that Tcf4 is one of the factors contributing to formation of differentially methylated regions. Conclusions: Our results reveal limited DNA methylation dynamics during small intestine stem cell differentiation and an impact of transcription factor binding on shaping the DNA methylation landscape during differentiation of stem cells in vivo

Chromosome 3p25.3 Gain Is Associated with Cisplatin Resistance and Is an Independent Predictor of Poor Outcome in Male Malignant Germ Cell Tumors

\ua9 American Society of Clinical Oncology. PURPOSE: Cisplatin is the main systemic treatment modality for male type II germ cell tumors (GCTs). Although generally very effective, 5%-10% of patients suffer from cisplatin-resistant disease. Identification of the driving mechanisms of resistance will enable improved risk stratification and development of alternative treatments. METHODS: We developed and characterized cisplatin-resistant GCT cell line models and compared their molecular characteristics with patient samples with cisplatin resistance and/or a poor clinical outcome. Subsequently, the association between the overlapping genetic features and clinical data was assessed. Finally, we used Cox regression to determine the prognostic relevance of these features within the currently used risk classification. RESULTS: Gain of chromosome 3p25.3 was detected in all cisplatin-resistant cell lines, and copy number of this region correlated with the level of resistance (R = 0.96, P = 1.5e-04). Gain of this region was detected at low frequencies in primary tumors and at higher frequencies in relapsed and/or cisplatin-resistant tumors. Chromosome 3p25.3 gain was associated with shorter progression-free survival and overall survival, with the strongest association observed in nonseminomas excluding pure teratomas. 3p25.3 gain was more frequently observed in tumors with yolk sac tumor histology and predicted adverse outcome independent of the International Germ Cell Cancer Collaborative Group risk classification and the presence of TP53/MDM2 alterations. CONCLUSION: On the basis of both in vitro analyses and clinical data, we found 3p25.3 to be strongly associated with cisplatin resistance and poor clinical outcome in male type II GCTs. Using genomic profiling, 3p25.3 status could help to improve risk stratification in male patients with type II GCT. Further characterization of this locus and underlying mechanisms of resistance is warranted to guide development of novel treatment approaches for cisplatin-resistant disease

Targeted Chromatin Capture (T2C): A novel high resolution high throughput method to detect genomic interactions and regulatory elements.

Background: Significant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort. Techniques like Hi-C provide a genome-wide analysis. However, it requires massive sequencing effort with considerable costs. Here we describe a new technique termed Targeted Chromatin Capture (T2C), to interrogate large selected regions of the genome. T2C provides an unbiased view of the spatial organization of selected loci at superior resolution (single restriction fragment resolution, from 2 to 6 kbp) at much lower costs than Hi-C due to the lower sequencing effort. Results: We applied T2C on well-known model regions, the mouse β-globin locus and the human H19/IGF2 locus. In both cases we identified all known chromatin interactions. Furthermore, we compared the human H19/IGF2 locus data obtained from different chromatin conformation capturing methods with T2C data. We observed the same compartmentalization of the locus, but at a much higher resolution (single restriction fragments vs. the common 40 kbp bins) and higher coverage. Moreover, we compared the β-globin locus in two different biological samples (mouse primary erythroid cells and mouse fetal brain), where it is either actively transcribed or not, to identify possible transcriptional dependent interactions. We identified the known interactions in the β-globin locus and the same topological domains in both mouse primary erythroid cells and in mouse fetal brain with the latter having fewer interactions probably due to the inactivity of the locus. Furthermore, we show that interactions due to the important chromatin proteins, Ldb1 and Ctcf, in both tissues can be analyzed easily to reveal their role on transcriptional interactions and genome folding. Conclusions: T2C is an efficient, easy, and affordable with high (restriction fragment) resolution tool to address both genome compartmentalization and chromatin-interaction networks for specific genomic regions at high resolution for both clinical and non-clinical research

Round Spermatid Injection Rescues Female Lethality of a Paternally Inherited Xist Deletion in Mouse

In mouse female preimplantation embryos, the paternal X chromosome (Xp) is silenced by imprinted X chromosome inactivation (iXCI). This requires production of the noncoding Xist RNA in cis, from the Xp. The Xist locus on the maternally inherited X chromosome (Xm) is refractory to activation due to the presence of an imprint. Paternal inheritance of an Xist deletion (XpΔXist) is embryonic lethal to female embryos, due to iXCI abolishment. Here, we circumvented the histone-to-protamine and protamine-to-histone transitions of the paternal genome, by fertilization of oocytes via injection of round spermatids (ROSI). This did not affect initiation of XCI in wild type female embryos. Surprisingly, ROSI using ΔXist round spermatids allowed survival of female embryos. This was accompanied by activation of the intact maternal Xist gene, initiated with delayed kinetics, around the morula stage, resulting in Xm silencing. Maternal Xist gene activation was not observed in ROSI-derived males. In addition, no Xist expression was detected in male and female morulas that developed from oocytes fertilized with mature ΔXist sperm. Finally, the expression of the X-encoded XCI-activator RNF12 was enhanced in both male (wild type) and female (wild type as well as XpΔXist) ROSI derived embryos, compared to in vivo fertilized embryos. Thus, high RNF12 levels may contribute to the specific activation of maternal Xist in XpΔXist female ROSI embryos, but upregulation of additional Xp derived factors and/or the specific epigenetic constitution of the round spermatid-derived Xp are expected to be more critical. These results illustrate the profound impact of a dysregulated paternal epigenome on embryo d

A crucial role for the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specification

Mammalian development is regulated by the interplay of tissue-specific and ubiquitously expressed transcription factors, such as Sp1. Sp1 knockout mice die in utero with multiple phenotypic aberrations, but the underlying molecular mechanism of this differentiation failure has been elusive. Here, we have used conditional knockout mice as well as the differentiation of mouse ES cells as a model with which to address this issue. To this end, we examine

The clonal relation of primary upper urinary tract urothelial carcinoma and paired urothelial carcinoma of the bladder

The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor-specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities

Patient-Derived Xenografts and Organoids Recapitulate Castration-Resistant Prostate Cancer with Sustained Androgen Receptor Signaling

Castration-resistant prostate cancer (CRPC) remains an incurable and lethal malignancy. The development of new CRPC treatment strategies is strongly impeded by the scarcity of representative, scalable and transferable preclinical models of advanced, androgen receptor (AR)-driven CRPC. Here, we present contemporary patient-derived xenografts (PDXs) and matching PDX-derived organoids (PDXOs) from CRPC patients who had undergone multiple lines of treatment. These models were comprehensively profiled at the morphologic, genomic ( n = 8) and transcriptomic levels ( n = 81). All are high-grade adenocarcinomas that exhibit copy number alterations and transcriptomic features representative of CRPC patient cohorts. We identified losses of PTEN and RB1, MYC amplifications, as well as genomic alterations in TP53 and in members of clinically actionable pathways such as AR, PI3K and DNA repair pathways. Importantly, the clinically observed continued reliance of CRPC tumors on AR signaling is preserved across the entire set of models, with AR amplification identified in four PDXs. We demonstrate that PDXs and PDXOs faithfully reflect donor tumors and mimic matching patient drug responses. In particular, our models predicted patient responses to subsequent treatments and captured sensitivities to previously received therapies. Collectively, these PDX-PDXO pairs constitute a reliable new resource for in-depth studies of treatment-induced, AR-driven resistance mechanisms. Moreover, PDXOs can be leveraged for large-scale tumor-specific drug response profiling critical for accelerating therapeutic advances in CRPC. </p