Trithorax group proteins: switching genes on and keeping them active

Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology

Histone modifications across the cell cycle in undifferentiated and differentiating mouse embryonic stem cells

The role of post translational histone modifications in stem cells has been of increasing interest in recent years, however, the heritability of histone modifications has not yet been determined, and as such their status as epigenetic remains in question. Here we have taken the novel approach of comparing the enrichment of histone modifications, across specific genes and how they are modulated through various phases of the cell cycle: in doing so we address this question of heritability from a new perspective. Highly dynamic fluctuations in the enrichment of histone modifications were observed across the cell cycle in embryonic stem cells. In cell cycle regulated genes the patterns of modification enrichment revealed an increase in active marks either pre-emptive or at the point of expression, indicative of highly dynamic regulation, not a stable heritable transmission, perhaps reflective of the plasticity of these cells. Following on from this embryonic stem cells were differentiated for seven days, allowing the enforcement of canonical cell cycle regulation and a more lineage specific transcription profile. At this point histone modifications displayed a variety of patterns including what appeared to be the stable and presumably heritable transmission of H3K4me3 and H3K27me3 across the cell cycle

Functional analysis of Zfp819 in pluripotency and embryonic development

Pluripotenz wird durch viele Stammzell-spezifische Transkriptionsfaktoren wie Oct3/4, Nanog und Sox2 sowie deren Funktion in ihrem regulatorischen Netzwerk etabliert und aufrechterhalten. Viele Studien haben gezeigt, wie diese Pluripotenz-assoziierten Faktoren ihre Zielgene regulieren. Dies geschieht durch die Interaktion mit bekannten und unbekannten Interaktionspartnern. In der vorliegenden Arbeit haben wir Zfp819 als einen neuen Pluripotenz-assoziierten Faktor beschrieben und dessen Funktion in pluripotenten Stammzellen untersucht. Im ersten Teil der vorliegenden Arbeit haben wir zwei cDNA-Banken für Yeast two Hybdrid (Y2H)-Assays aus unterschiedlichen pluripotenten Stammzelltypen generiert. Dies hatte zum Ziel, potentielle Interaktionspartner eines Kandidatenproteins zu identifizieren um dadurch Eindrücke über die Funktion des Proteins zu gewinnen. Für die Identifizierung von potentiellen Interaktionspartnern von Zfp819 haben wir die cDNA-Bank aus embryonalen Stammzellen benutzt. Wir konnten 17 putative Interaktionspartner identifizieren und daraus ein hypothetisches „Interaktom“ von Zfp819 generieren. Die Einordnung der putativen Interaktionspartner nach ihrer Gen-Ontologie (GO) ließ vermuten, dass Zfp819 eine Rolle in der Regulation der Transkription, der Aufrechterhaltung der genetischen Integrität und im Zellzyklus bzw. bei der Apoptose spielt. Im zweiten Teil der vorliegenden Arbeit wurde die sehr intensive Expression von Zfp819 in undifferenzierten pluripotenten Zelllinien gezeigt. Desweiteren konnte die Promotorregion von Zfp819 identifiziert werden, und es wurde gezeigt, dass diese mit epigenetischen Mustern ausgestattet ist. Zusätzlich konnten wir Regionen im Zfp819-Gen identifizieren, die für die nukleäre Lokalisation von Zfp819 verantwortlich sind. Desweiteren konnten wir zeigen, dass Zfp819 in der transkriptionellen Repression von spezifischen endogenen, retroviralen Elementen (ERVs) in pluripotenten Zellen eine Rolle spielt. Durch zelluläre und biochemische Studien konnten wir zeigen, dass Zfp819 mit vielen Proteinen interagiert (z.B. Kap1 und Chd4), welche für die Aufrechterhaltung der genomischen Integrität von Bedeutung sind. Tatsächlich resultierte der Verlust von Zfp819 in embryonalen Stammzellen in einer erhöhten Anfälligkeit für DNA-Schäden und in einer verminderten DNA-Reparatur. Zusammenfassend lassen die Identifizierung der Interaktionspartner sowie die Ergebnisse der molekularen und der funktionellen Studien vermuten, dass Zfp819 durch die Unterdrückung von ausgewählten ERVs eine Rolle in der Regulation der genomischen Stabilität von pluripotenten Zellen spielt

G1 Dynamics at the Crossroads of Pluripotency and Cancer

G1 cell cycle phase dynamics are regulated by intricate networks involving cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors, which control G1 progression and ensure proper cell cycle transitions. Moreover, adequate origin licensing in G1 phase, the first committed step of DNA replication in the subsequent S phase, is essential to maintain genome integrity. In this review, we highlight the intriguing parallels and disparities in G1 dynamics between stem cells and cancer cells, focusing on their regulatory mechanisms and functional outcomes. Notably, SOX2, OCT4, KLF4, and the pluripotency reprogramming facilitator c-MYC, known for their role in establishing and maintaining stem cell pluripotency, are also aberrantly expressed in certain cancer cells. In this review, we discuss recent advances in understanding the regulatory role of these pluripotency factors in G1 dynamics in the context of stem cells and cancer cells, which may offer new insights into the interconnections between pluripotency and tumorigenesis

Mechanisms of cohesin mediated regulation of gene expression

The cohesin complex is essential for proper sister chromatid segregation during cell division and post-replicative DNA repair. Cohesin is also important for the regulation of gene expression. However, the mechanisms by which cohesin impacts gene expression remain incompletely understood. Owing to its vital role in cell division and DNA repair, loss of cohesin can indirectly impact gene expression programme in cycling cells. Thus, in order to investigate cohesin’s role in gene regulation, a conditional knockout system was used which allowed rapid depletion of the cohesin subunit RAD21 and avoided secondary stress-induced effects on gene expression. Acute depletion of cohesin in mouse embryonic stem (ES) cells did not lead to a global collapse in pluripotency. Instead, the impact of cohesin depletion was limited to about 600 genes and was locus-specific in terms of direction of deregulation. A subset of deregulated genes was selected based on the positioning of cis-regulatory elements and relevance to the pluripotent state and the role of cohesin in mediating long-range interactions was analysed using chromosome conformation capture (3C). Interestingly, cohesin binding, DNA looping and transcriptional changes were not always correlated. At some of the loci tested, these interactions were maintained after removal of cohesin, questioning models where cohesin regulates gene expression solely by mediating long-range interactions. One of the pluripotency factors affected by cohesin depletion in ES cells was Myc. Experiments analysing the expression of Myc showed that it was post-transcriptionally upregulated, specifically in cohesin deficient ES cells growing in defined media supplemented with ERK and GSK3β inhibitors (2i media). Further investigation revealed that contrary to the previously reported downregulation of Myc upon cohesin depletion, cohesin was not essential for Myc expression in various cell types. In separate experiments, I investigated if cohesin was required for the transcriptional activation of a silent gene in response to extracellular stimuli. Results from IFNγ induction of cohesin deficient non-cycling mouse embryonic fibroblasts (MEFs) showed that cohesin was important for the activation of MHC class II genes and their master regulator Ciita. The expression of MHC class I genes and the associated regulatory factors remained unaffected by cohesin depletion. Further evidence is provided for the involvement of cohesin in regulating transcription by modulating RNA polymerase processivity and through the action of PTIP subunit of the MLL complexes. Altogether my work gives an insight into the role of cohesin in mediating long-range DNA interactions important for regulation of gene expression and explores novel mechanisms of gene activation by cohesin.Open Acces

Characterization of a dand5 p.r152h control-derived ips cell line to be used as a tool for heart disease modeling

RESUMO: As doenças cardíacas congênitas (DCC) e os defeitos de lateralidade associados são uma das principais preocupações de saúde e 1,35 milhões de pessoas são diagnosticadas com DCC a cada ano em todo o mundo. A complexidade do desenvolvimento do coração e os obstáculos para investigar os fenótipos da doença tornam difícil identificar as causas subjacentes das DCC. O gene DAND5 é o homólogo humano do Cerl-2 de camundongo que codifica uma proteína envolvida na regulação da via de sinalização Nodal antagonizando a proteína Nodal no nódulo e inibindo a sinalização Nodal na mesoderme da placa lateral direita (RLPM). Em um estudo anterior, uma nova variante não-sinônima c.455G> A da DAND5 heterozigótica foi identificada e vinculada ao risco de doença em dois pacientes com DCC decorrentes de defeitos de lateralidade. Para modelar o fenótipo dos pacientes com a variante c.455G> A, uma linha iPSC humana foi previamente gerada e caracterizada (Cristo et al., 2017). Neste trabalho, caracterizamos uma linha iPSC de controle DAND5 de um doador do sexo masculino sadio (sem a variante) para servir de controle para a linha DAND5 c.455G>A. A caracterização foi baseada na detecção de marcadores de pluripotência a nível de genes e proteínas, e potencial diferencial in vitro. A análise de repetições curtas em tandem (STR) usou para provar a identidade genética das células ERE e das iPSCs reprogramadas. A cariotipagem das iPSCs após ≥ 20 passagens mostrou a estabilidade das iPSCs altamente proliferativas. Além disso, o teste de detecção de Mycoplasma mostrou a esterilidade da cultura de células. Esta linha celular de controlo será comparada com a linhagem celular c.455G> A da variante DAND5, para explorar ainda mais o nosso conhecimento sobre as consequências da variante nos fenótipos dos doentes (modelação da doença) e, mais precisamente, na proliferação de cardiomiócitos (terapia possível).ABSTRACT: Congenital heart diseases (CHDs) and associated laterality defects are a major health concern and 1,35 million people are diagnosed with CHDs each year worldwide. The complexity of heart development and the hurdles to investigate the disease phenotypes make it challenging to identify the underlying causes of CHDs. DAND5 is the human homologue of mouse Cerl-2 gene that codes for a protein involved in regulating the Nodal signalling pathway by antagonizing the Nodal protein in the node and inhibiting the Nodal signaling in the right lateral plate mesoderm (R-LPM). In a previous study, a new DAND5 heterozygous nonsynonymous variant c.455G>A was identified and linked to the risk of disease in two patients with CHDs arising from laterality defects. In order to model the phenotype of the patients with the variant c.455G>A, a human iPSC line was previously generated and characterized (Cristo et al., 2017). In this work, we characterized a DAND5-control iPSC line from a healthy male donor (without the variant) to serve as control for the DAND5 c.455G>A line. The characterization was based on the detection of pluripotency markers at gene- and protein level, and in vitro differential potential. Short tandem repeats (STR) analysis has used to prove the genetic identity of the ERE cells and the reprogrammed iPSCs. Karyotyping of the iPSCs after ≥20 passages has shown the stability of the highly proliferating iPSCs. Additionally, Mycoplasma detection test showed the sterility of the cell culture. This control cell line will be compared to the DAND5 variant c.455G>A cell line to further exploit our knowledge on the consequences of the variant in the phenotypes of the patients (disease modelling) and more precisely in the modulation of cardiomyocyte proliferation (possible therapy)

Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines

To overcome loss of stem-like properties and spontaneous differentiation those hinder the expansion and application of human mesenchymal stem cells (hMSCs), we have clonally isolated permanent and stable human MSC lines by ectopic overexpression of primary cell cultures of hMSCs with HPV 16 E6E7 and human telomerase reverse transcriptase (hTERT) genes. These cells were found to have a differentiation potential far beyond the ordinary hMSCs. They expressed trophoectoderm and germline specific markers upon differentiation with BMP4 and retinoic acid, respectively. Furthermore, they displayed higher osteogenic and neural differentiation efficiency than primary hMSCs or hMSCs expressed HPV16 E6E7 alone with a decrease in methylation level as proven by a global CpG island methylation profile analysis. Notably, the demethylated CpG islands were highly associated with development and differentiation associated genes. Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells. These data demonstrate these cells not only are a useful tool for the studies of cell differentiation both for the mesenchymal and neurogenic lineages, but also provide a valuable source of cells for cell therapy studies in animal models of skeletal and neurological disorders

Regulation of Pluripotency and Differentiation by Chromatin Remodeling Factors

Central to the control of virtually all cellular activity is the regulation of gene expression. In eukaryotes, this regulation is greatly influenced by chromatin structure, which is itself regulated by numerous chromatin-remodeling complexes. These are typically large protein complexes with interchangeable subunits that allow for highly specialized functions in different cell types. Moreover, additional specificity can be gained through complexes formed from different subunit isoforms. Histone modifications also regulate chromatin by recruiting remodeling complexes to particular genomic regions. In this thesis we characterize MBD3C, an isoform of the Nucleosome Remodeling and Deacetylase (NuRD) complex subunit MBD3. MBD3 is essential for pluripotency and development, but MBD3C appears to be expressed only in embryonic stem cells (ESCs), and whether it forms a distinct NuRD complex, how its expression is regulated, and its precise function(s) remain unknown. We show that MBD3C forms a complete NuRD complex that functions redundantly with the other MBD3 isoforms in ESC gene regulation. Furthermore, MBD3C binds the SET/MLL complex subunit WDR5 through a conserved motif within its unique N-terminal region, and this interaction is necessary for the regulation of \u3e2,000 ESC genes. Together, these findings indicate that ESCs can utilize isoforms of the same protein to achieve similar functions through diverse mechanisms. The second part of this thesis focuses on the role of the histone modification H3.3K56ac in pluripotency and differentiation. Although H3K56ac is well-studied in yeast, in mammalian cells it is far less abundant and its functions are largely unknown. Our data indicate that the H3.3K56R mutant is largely normal for ESC maintenance and loss of pluripotency markers during differentiation, but H3.3K56ac is necessary for proper lineage commitment. Ongoing studies will characterize the H3.3K56Q phospho-mimetic mutant during differentiation, and examine H3.3K56ac function at lineage-specific genes