Disruption of FEM1C-W gene in zebra finch: evolutionary insights on avian ZW genes

Sex chromosome genes control sex determination and differentiation, but the mechanisms of sex determination in birds are unknown. In this study, we analyzed the gene FEM1C which is highly conserved from Caenorhabditis elegans to higher vertebrates and interacts with the sex determining pathway in C. elegans. We found that FEM1C is located on the Z and W chromosome of zebra finches and probably other Passerine birds, but shows only Z linkage in other avian orders. In the zebra finch, FEM1C-W is degraded because of a point mutation and possibly because of loss of the first exon containing the start methionine. Thus, FEM1C-W appears to have degenerated or been lost from most bird species. FEM1C-Z is expressed in a cytoplasmic location in zebra finch fibroblast cells, as in C. elegans. FEM1C represents an interesting example of evolutionary degradation of a W chromosome gene

Histone H4 deacetylation plays a critical role in early gene silencing during neuronal apoptosis

<p>Abstract</p> <p>Background</p> <p>Silencing of normal gene expression occurs early in the apoptosis of neurons, well before the cell is committed to the death pathway, and has been extensively characterized in injured retinal ganglion cells. The causative mechanism of this widespread change in gene expression is unknown. We investigated whether an epigenetic change in active chromatin, specifically histone H4 deacetylation, was an underlying mechanism of gene silencing in apoptotic retinal ganglion cells (RGCs) following an acute injury to the optic nerve.</p> <p>Results</p> <p>Histone deacetylase 3 (HDAC3) translocates to the nuclei of dying cells shortly after lesion of the optic nerve and is associated with an increase in nuclear HDAC activity and widespread histone deacetylation. H4 in promoters of representative genes was rapidly and indiscriminately deacetylated, regardless of the gene examined. As apoptosis progressed, H4 of silenced genes remained deacetylated, while H4 of newly activated genes regained, or even increased, its acetylated state. Inhibition of retinal HDAC activity with trichostatin A (TSA) was able to both preserve the expression of a representative RGC-specific gene and attenuate cell loss in response to optic nerve damage.</p> <p>Conclusions</p> <p>These data indicate that histone deacetylation plays a central role in transcriptional dysregulation in dying RGCs. The data also suggests that HDAC3, in particular, may feature heavily in apoptotic gene silencing.</p

Proteomic Profiles of Mesenchymal Stem Cells Induced by a Liver Differentiation Protocol

The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs

Karyotypic polymorphism of the zebra finch Z chromosome

We describe a karyotypic polymorphism on the zebra finch Z chromosome. This polymorphism was discovered because of a difference in the position of the centromere and because it occurs at varying frequencies in domesticated colonies in the USA and Germany and among two zebra finch subspecies. Using DNA fluorescent in situ hybridization to map specific Z genes and measurements of DNA replication, we show that this polymorphism is the result of a large pericentric inversion involving the majority of the chromosome. We sequenced a likely breakpoint for the inversion and found many repetitive sequences. Around the breakpoint, there are numerous repetitive sequences and several copies of PAK3 (p21-activated kinase 3)-related sequences (PAK3Z) which showed testes-specific expression by RT-PCR. Our findings further suggest that the sequenced genome of the zebra finch may be derived from a male heterozygote for the Z chromosome polymorphism. This finding, in combination with regional differences in the frequency of the polymorphism, has important consequences for future studies using zebra finches

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small and a facile technology to characterize them is lacking. Using a strategy combining Global Protein Stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Using CRISPR screening, we established that the stability of many proteins is regulated through degrons located at their C-terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complexes adaptors that regulate C-terminal degrons including six CRL2 and two CRL4 complexes and computationally implicate multiple non-CRLs in end recognition. Human proteome analysis revealed that the C-termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3 ligase-dependent modulation of proteome composition. Thus, we propose that a series of ‘C-end rules’ operate to govern protein stability and shape the eukaryotic proteome

Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is variable and asymmetric

BACKGROUND: Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the βGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice. RESULTS: Optic nerve degeneration, characterized by axon loss and gliosis is first apparent in mice between 8 and 9 months of age. Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe. Although nerve damage is typically bilateral, the progression of disease is asymmetric between the eyes of individual mice. Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region. Ganglion cell loss, as a function of the loss of βGeo expression, is evident in some mice between 8 and 10 months of age and is prevalent in the majority of mice older than 10.5 months. Most eyes display a uniform loss of ganglion cells throughout the retina, but many younger mice exhibit focal loss of cells in sectors extending from the optic nerve head to the retinal periphery. Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal. CONCLUSION: A comparison of the data collected from the two cohorts of mice used for this study suggests that the initial site of damage in this disease is to the axons in the optic nerve, followed by the subsequent death of the ganglion cell soma

The structure and regulation of Cullin 2 based E3 ubiquitin ligases and their biological functions.

BACKGROUND: Cullin-RING E3 ubiquitin ligase complexes play a central role in targeting cellular proteins for ubiquitination-dependent protein turnover through 26S proteasome. Cullin-2 is a member of the Cullin family, and it serves as a scaffold protein for Elongin B and C, Rbx1 and various substrate recognition receptors to form E3 ubiquitin ligases. MAIN BODY OF THE ABSTRACT: First, the composition, structure and the regulation of Cullin-2 based E3 ubiquitin ligases were introduced. Then the targets, the biological functions of complexes that use VHL, Lrr-1, Fem1b, Prame, Zyg-11, BAF250, Rack1 as substrate targeting subunits were described, and their involvement in diseases was discussed. A small molecule inhibitor of Cullins as a potential anti-cancer drug was introduced. Furthermore, proteins with VHL box that might bind to Cullin-2 were described. Finally, how different viral proteins form E3 ubiquitin ligase complexes with Cullin-2 to counter host viral defense were explained. CONCLUSIONS: Cullin-2 based E3 ubiquitin ligases, using many different substrate recognition receptors, recognize a number of substrates and regulate their protein stability. These complexes play critical roles in biological processes and diseases such as cancer, germline differentiation and viral defense. Through the better understanding of their biology, we can devise and develop new therapeutic strategies to treat cancers, inherited diseases and viral infections