Faculty Bibliography

Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by Phf5a in stem cells, which directs their transcriptional programme, ultimately regulating maintenance of pluripotency and cellular reprogramming.

Successful pregnancy requires delicate control over the immunological and inflammatory properties of the maternal/fetal interface. For example, inflammation within the pregnant uterus is likely to be a major instigator of preterm birth, while inadequate immune surveillance of the maternal/fetal interface likely increases the risk of fetal and placental infection. We will discuss our recent work on the molecular and cellular pathways that regulate immune cell trafficking and inflammation within the pregnant mouse uterus. This work points to the seminal importance of the decidua, i.e. the specialized endometrial stromal tissue that encases the implanted embryo, and in particular an epigenetic program active in decidual stromal cells that we previously found transcriptionally silences the expression of chemokine genes that control effector T cell trafficking. Our recent results suggest that this program also silences a multitude of other important genes, including ones whose misexpression might be expected to generate uterine inflammation and lead to a variety of pregnancy complications including preterm birth

Cell-specific gene expression programs rely on the orchestrated function of transcription factors, co-activators, the transcriptional machinery and non-coding RNA elements at enhancer and promoter elements. Mediator is a large co-activator complex that bridges promoters with transcription factors bound to cell-specific enhancers. Here we describe Med12, a member of the Mediator complex kinase module, as an indispensable regulator of hematopoietic stem cell (HSC) maintenance and differentiation. Conditional deletion of Med12 rapidly exhausts adult bone marrow HSCs leading to lethality. Conversely, HSC-specific deletion of other members of the Mediator complex kinase module, Med13, CyclinC and CDK8 are dispensable for HSC function. We also show that Med12 is an enhancer element that co-localizes with essential hematopoietic transcription factors and enhancer-specific histone marks. In this context, we demonstrate that Med12 loss in HSCs results in rapid dysregulation of stemness signatures, as well as failure of enhancer activation by depletion of H3K27Ac. Finally, we find that Med12 promotes stabilization of p300 at enhancers, thereby contributing to the preservation of HSC homeostasis. Collectively, our data describes a novel, kinase-independent function for Med12 in the regulation of HCS enhancer elements and the maintenance of normal stem cell function. As a result, we believe that alterations in the Med12-dependent regulation of HSC enhancers may contribute to malignant transformation and disease

Study question: What do RNA seq-profiles tell us about gene activity in preimplantation embryos known to be normal vs. trisomic for chromosomes 21, 18, 15 and 22. Summary answer: RNA-Seq profiles are karyotype-dependent and appear to correlate with viability. X and Y transcription is surprisingly active at the preimplantation stage. What is known already: Embryonic karyotypic abnormalities are the most common cause of implantation failure and miscarriage. Some such embryos can progress to viability as evidenced by the birth of children with Trisomy 21, 18 and 15. An abnormal karyotype dictates developmental aberrations, and for these differences to affect phenotype, variations in gene expression must occur in the developing embryo. We set out to determine if such changes could be identified in embryos as early as the pre-implantation stage. Study design, size, duration: We used a cohort of 19 pre-implantation embryos (day 5 and 6 blastocysts); three being normal, five trisomy 15, two trisomy 22, three trisomy 21 and three trisomy 18, and three of unknown karyotype. Participants/materials, setting, methods: After written consent, analysis was performed on high quality embryos that previously underwent trophectoderm biopsy with array comparative genomic hybridization (aCGH) or next generation sequencing (NGS) for 24-chromosome aneuploidy screening and vitrified. Blastocysts were lysed immediately after thawing, and cDNA was synthesized and amplified, followed by RNA-Seq library preparation for deep Illuminabased sequencing. Sex and chromosomal aneuploidy were used as parameters for comparative analysis using a bioinformatics pipeline using a sensitivity of 0.05. Main results and the role of chance: Principal Component analysis (PCA) revealed that normal embryos clustered in proximity to trisomies 21 and 18, while 15 and 22 clustered separately. This suggested that early gene expression may correlate with viability. PCA did not distinguish between male and female embryos. Differential gene expression was calculated using DESEQ2, an R package that estimates the variance-mean dependence and tests for differential expression using a model using the negative binomial distribution. A comparison of sex-specific gene expression showed that the top differentially expressed genes were on the sex chromosomes, including various Y-linked transcription factors, helicases, and ribosomal proteins, as well as a testis-specific regulatory transcript, and >200 X-chromosome genes. Trisomy 21 embryos were the closest to normal embryos, with only 3 genes on chromosome 21 expressed more highly in the trisomic embryos. In contrast, trisomy 22 embryos (non viable), had 684 differentially expressed genes, 32 of which on chromosome 22. Trisomy 18 embryos had only one highly expressed significant gene, and it is located on chromosome 18. Trisomy 15 embryos had 829 differentially expressed genes, of which 83 were in chromosome 15. Our results suggest that the less viable trisomies have bigger gene expression differences, even at this very early stage. Limitations, reasons for caution: While the karyotypes were analyzed by stringent methods, embryos may have had elements of mosaicism or chromosomal structural abnormalities. The genes identified by RNA-Seq need to be validated using orthogonal methods. Wider implications of the findings: We have expanded the knowledge of the transcriptome of the human pre-implantation embryo as they relate to aneuploidy and sex. This information can now be used to further our understanding of early embryonic development and stem cell biology, and to identify biomarkers for non-invasive preimplantation genomic screening

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs). To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor beta (TGF-beta) together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-beta inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-beta/mitogen-activated protein (MAP) kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells.

Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.