Mechanisms of RNF168 nucleosome recognition and ubiquitylation.

RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear.

Preliminary Study on Expression and Function of the Chicken W Chromosome Gene in Embryonic Gonads.

The sex chromosomes of birds are designated Z and W. The male is homogamous (ZZ), and the female is heterogamous (ZW). The chicken W chromosome is a degenerate version of the Z chromosome and harbors only 28 protein-coding genes.

A stress-induced cilium-to-PML-NB route drives senescence initiation.

Cellular senescence contributes to tissue homeostasis and age-related pathologies. However, how senescence is initiated in stressed cells remains vague. Here, we discover that exposure to irradiation, oxidative or inflammatory stressors induces transient biogenesis of primary cilia, which are then used by stressed cells to communicate with the promyelocytic leukemia nuclear bodies (PML-NBs) to initiate senescence responses in human cells.

Avian Sex Determination: A Chicken and Egg Conundrum.

Background: Primary sex determination is the developmental process that results in the sexual differentiation of the gonads. Vertebrate sex determination is generally considered to follow the model based on the mammalian system, where a sex-specific master regulatory gene activates one of the two different gene networks that underlie testis and ovary differentiation.

Summary: It is now known that, while many of the molecular components of these pathways are conserved across different vertebrates, a wide variety of different trigger factors are utilized to initiate primary sex determination.

Sexually dimorphic expression of a chicken sex chromosome gene (VCP) reflects differences in gonadal development between males and females.

The chicken has a Z-W sex chromosome system, in which the males are the homogametic sex (ZZ) and the females the heterogametic sex (ZW). The smaller W chromosome is generally considered to be a highly degraded copy of the Z chromosome that retains around 28-30 homologous protein-coding genes' These Z-W homologues are thought to have important, but undefined, roles in development, and here we explore the role of one of these genes, VCP (Valosin Containing Protein) in gonadogenesis. We established RNA expression levels of both Z and W VCP homologues, the levels of VCP protein, and the cellular localization of VCP protein in male and female embryonic gonads during development.

Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.

The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown.

Expression Profile of Chicken Sex Chromosome Gene BTF3 is Linked to Gonadal Phenotype.

In birds, the female is heterogametic (ZW) and the male homogametic (ZZ). The small W chromosome comprises only 28 protein coding genes (homologues to Z chromosome counterparts) and a number of repeat regions. Here, we report our analysis of one of these genes, BTF3 (basic transcription factor 3), which exhibits differential expression during gonadogenesis.

Oestrogen in the chick embryo can induce chromosomally male ZZ left gonad epithelial cells to form an ovarian cortex that can support oogenesis.

In chickens, the embryonic ovary differentiates into two distinct domains before meiosis: a steroidogenic core (the female medulla), overlain by the germ cell niche (the cortex). The differentiation of the medulla is a cell-autonomous process based on chromosomal sex identity (CASI). In order to address the extent to which cortex differentiation depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering oestrogen levels of ZW and ZZ embryos.

Feather arrays are patterned by interacting signalling and cell density waves.

Feathers are arranged in a precise pattern in avian skin. They first arise during development in a row along the dorsal midline, with rows of new feather buds added sequentially in a spreading wave. We show that the patterning of feathers relies on coupled fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling together with mesenchymal cell movement, acting in a coordinated reaction-diffusion-taxis system.

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR.

Polo-like kinase 1 coordinates biosynthesis during cell cycle progression by directly activating pentose phosphate pathway.

Two hallmarks for cancer cells are the accelerated cell cycle progression as well as the altered metabolism, however, how these changes are coordinated to optimize the growth advantage for cancer cells are still poorly understood. Here we identify that Polo-like kinase 1 (Plk1), a key regulator for cell mitosis, plays a critical role for biosynthesis in cancer cells through activating pentose phosphate pathway (PPP). We find that Plk1 interacts with and directly phosphorylates glucose-6-phosphate dehydrogenase (G6PD).

Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18.

The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechanisms.

Structural and biochemical insights into the DNA-binding mode of MjSpt4p:Spt5 complex at the exit tunnel of RNAPII.

Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription.

Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages.

We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages.

Structural investigation of the interaction between the tandem SH3 domains of c-Cbl-associated protein and vinculin.

c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin.

Gonadal asymmetry and sex determination in birds.

Although vertebrates display a superficial bilateral symmetry, most internal organs develop and locate with a consistent left:right asymmetry. There is still considerable debate as to when this process actually begins, but it seems that, at least for some species, the initial steps occur at a very early stage of development. In recent years, a number of model systems, including the chick embryo, have been utilised to increase our understanding of the molecular basis of this complex developmental process.

Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research, drug discovery, and the treatment of liver diseases holds great promise. Here we describe a three-stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide, followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation, hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation, expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially.

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines.

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture.

Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities.

The utilization of human hepatocytes for biomedical research, drug discovery, and treatment of liver diseases is hindered by the limited availability of donated livers and the variability of their derived hepatocytes. Human embryonic stem cells (hESCs) are pluripotent and provide a unique, unlimited resource for human hepatocytes. However, differentiation of hESCs to hepatocytes remains a challenge.

Blood vessel/epicardial substance (bves) expression, essential for embryonic development, is down regulated by Grk/EFGR signalling.

The Pop1/Bves (blood vessel/epicardial substance) gene is a member of the popeye gene family recently identified in various species. It encodes a potential transmembrane glycoprotein and is a cell adhesion molecule present in skeletal and cardiac muscle and epithelia. We isolated the Drosophila homologue of Bves (DmBves) and found, using in situ hybridisation to RNA in ovaries, that bves is expressed in all follicular epithelial cells surrounding the oocyte at stage 10, except those in very posterior and anterior-dorsal regions adjacent to the oocyte.

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes.

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor.