Lack of Rybp in Mouse Embryonic Stem Cells Impairs Cardiac Differentiation

Abstract

Ring1 and Yy1 Binding Protein (Rybp) has been implicated in transcriptional regulation, apoptotic signaling and as a member of the polycomb repressive complex 1 has important function in regulating pluripotency and differentiation of embryonic stem cells. Earlier, we have proven that Rybp plays essential role in mouse embryonic and central nervous system development. This work identifies Rybp, as a critical regulator of heart development. Rybp is readily detectable in the developing mouse heart from day 8.5 of embryonic development.Prominent Rybp expression persists during all embryonic stages and Rybp marks differentiated cell types of the heart. By utilizing rybp null embryonic stem cells (ESCs) in an in vitro cardiac differentiation assay we found that rybp null ESCs do not form rhythmically beating cardiomyocytes. Gene expression profiles revealed a down-regulation of terminal cardiac and upregulation of germ line specific markers in the rybp null cardiomyocytes. Furthermore, transcriptome analysis uncovered a number of novel candidate target genes regulated by Rybp.Among these are several important in cardiac development and contractility such as Plagl1, Isl1, This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 2Tnnt2. Importantly, forced expression of rybp in rybp deficient ESCs by a lentiviral vector was able to rescue the mutant phenotype. Our data provides evidence for a previously unrecognized function of Rybp in heart development.and pointing out the importance of germ cell lineage gene silencing during somatic differentiation. IntroductionA complex network of transcription factors governs the temporal and spatial patterns of gene expression in the organs of the developing embryo proper. Transcription factors also have important roles in postnatal and adult life in maintaining the pattern of differentiated gene expression [1][2][3]. Ring1 and Yy1 binding protein (Rybp; or also known as Dedaf (Death Effector Domain Associated Factor), UniGene Mm.321633; MGI:1929059) is a repressor protein that is also a member of the mammalian polycomb repressive complex 1 (PRC1) [4]. Rybp first was described as binding partner for the polycomb group protein (PcG) Ring1A (Ring1; ortholog of Drosophila dRing/Sce) and also was shown to associate with Ring1B (Ring2/Rnf2; ortholog of Drosophila dRing/Sce) and M33 (Pc1; ortholog of Drosophila Pc), components of the PRC1 multiprotein complex [5]. Our previous work showed that Rybp is selectively upregulated in distinct structures and cell types of the developing eye and the central nervous system (CNS) and, may also play a role in the development of more mature neurons [6,7]. We have also shown that rybp is essential for the development of the mouse embryo proper and that homozygous null mouse embryos cannot develop further implantation (embryonic day 5.5 (E5.5)). Notably, in a subset of heterozygous animals and in rybp −/− ↔rybp +/+ chimeras, alt...

From Flies to Mice: The Emerging Role of Non-Canonical PRC1 Members in Mammalian Development

“…Parallel in vitro studies also showed that Rybp has important role in differentiation of stem cells. Whilst the Rybp −/− ES cells are viable, can proliferate and can be maintained for an unlimited time, they do not form contractile cardiomyocytes (CMCs) ( Table 2) [244], and form less matured neurons, astrocytes and oligodendrocytes in vitro (Table 2) [245]. These results are in agreement with previous in vivo studies suggesting the role of Rybp in differentiation rather than self-renewal [166,240].…”

“…The majority of these mutations were generated decades ago [148], before the discovery of the canonical and non-canonical PRC1 complexes. Gene ablation of the non-canonical RYBP subunit results peri-implantational lethality in mice [166] and ES cells have compromised differentiation abilities [244,245]. Gene ablation of two ncPRC1.1 members, Usp7 [294] and Kdm2b [280], or the ncPRC1.6 member l3mbtl2, is also embryonic lethal [215], which highlights their essential function in embryogenesis.…”

Rybp is required for neural differentiation of mouse embryonic stem cells

“…In our experimental system expression of mesodermal marker Gsc was increased 3x at d7 in both cell lines ( Figure 11A) and there was no difference in Gsc expression between the cell lines. This coincides with our previous observations when during in vitro cardiac differentiation both early mesodermal markers Gsc and Brachyury were expressed as expected but the rybp -/-cells were not able to produce contractible cardiomyocytes (Ujhelly et al, 2015). This result shows that the lack of Rybp influences differentiation at later stages rather then mesoderm formation.…”

Multiple roles of Ring 1 and YY1 binding protein in physiology and disease

“…By constructing four RYBP mouse models, this team also showed that dysregulated RYBP expression resulted in retinal coloboma, malformed lenses, defects in anterior eye development and corneal neovascularization, indicating that RYBP plays critical roles in mouse eye development [12]. Additionally, Ujhelly et al suggested that RYBP is also important for both cardiac and germ cell development [13]. In terms of cardiac development, the absence of RYBP in ESCs blocked cardiac differentiation to contractile cardiomyocytes, possibly through regulation of the expression of Plagl1, Isl1 and Tnnt2 genes.…”

“…In terms of cardiac development, the absence of RYBP in ESCs blocked cardiac differentiation to contractile cardiomyocytes, possibly through regulation of the expression of Plagl1, Isl1 and Tnnt2 genes. Furthermore, these impaired phenotypes were rescued by ectopic expression of RYBP using a lentivirus vector [13]. In contrast to the active function in development, Zhou et al found that the expression of RYBP and its binding protein YY1 were gradually decreased during C2C12 myoblast differentiation, accompanied by miR-29 overexpression, indicating that RYBP acts as a repressor of skeletal myogenesis [14].…”