Üsküdar mesireleri

Taha Toros Arşivi, Dosya No: 63-Salacak-Üsküdarİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

ROCK-Dependent Phosphorylation of NUP62 Regulates p63 Nuclear Transport and Squamous Cell Carcinoma Proliferation

p63, more specifically its DNp63a isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to main- tain the undifferentiated status is associated with defects in DNp63a nuclear transport. We further find that differentiation- inducible Rho kinase reduces the interaction between NUP62 and DNp63a by phosphorylation of phenylalanine–glycine regions of NUP62, attenuating DNp63a nuclear import. Our results character- ize NUP62 as a gatekeeper for DNp63a and uncover its role in the control of cell fate through regulation of DNp63a nuclear transport in SCC

Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution

Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage

UBE3A-mediated regulation of imprinted genes and epigenome-wide marks in human neurons.

The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition

UBE3A-mediated regulation of imprinted genes and epigenome-wide marks in human neurons

<p>The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including <i>UBE3A</i>, which is maternally expressed exclusively in neurons. <i>UBE3A</i> encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of <i>UBE3A</i> in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition.</p

ROCK‐dependent phosphorylation of NUP62 regulates p63 nuclear transport and squamous cell carcinoma proliferation

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation-inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine-glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC

Cumulative Impact of Polychlorinated Biphenyl and Large Chromosomal Duplications on DNA Methylation, Chromatin, and Expression of Autism Candidate Genes.

Rare variants enriched for functions in chromatin regulation and neuronal synapses have been linked to autism. How chromatin and DNA methylation interact with environmental exposures at synaptic genes in autism etiologies is currently unclear. Using&nbsp;whole-genome bisulfite sequencing in brain tissue and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, we find that significant global DNA hypomethylation is enriched over autism candidate genes and affects gene expression. The cumulative effect of multiple chromosomal duplications and exposure to the pervasive persistent organic pollutant PCB 95 altered methylation of&nbsp;more than 1,000 genes. Hypomethylated genes were enriched for H2A.Z, increased maternal UBE3A in Dup15q corresponded to reduced levels of RING1B, and bivalently modified H2A.Z was altered by PCB 95 and duplication. These results demonstrate the compounding effects of genetic and environmental insults on the neuronal methylome that converge upon dysregulation of chromatin and synaptic genes

Scoliosis in transgenic founder zebrafish expressing <i>lbx1b</i> using the <i>GATA2</i> minimal promoter and the <i>lbx1b</i> enhancer.

<p>(A) The transgene construct is shown. The <i>GATA2</i> minimal promoter and <i>lbx1b</i> enhancer cooperatively drive <i>lbx1b</i> expression. (B) Dorsal views of live <i>GATA2-1b</i>:<i>MCS</i> (ctrl) or <i>GATA2-1b</i>:<i>lbx1b</i> (<i>lbx1b</i>) injected embryos with 10–13 somites. Somite arrangement is bilateral and symmetric in control embryos, but asymmetrical in <i>lbx1b</i> embryos. The yellow arrows indicate the mediolateral length of somites. (C) Dorsal views of 6 dpf larvae. The red and blue dotted lines indicate the boundaries of the dorsal melanophore stripes and the trunk, respectively. A displaced dorsal melanophore stripe was observed in <i>lbx1b</i> larvae. (D) Lateral views of alizarin red-stained larvae at 21 dpf. The white and yellow arrows indicate the hemivertebrae and block vertebra that developed from the deformed notochord in <i>lbx1b</i> larva, respectively. (E) Dorsal views and micro-computed tomography (μCT) analysis of adult zebrafish. Scoliosis was observed in adult fish grown from embryos with mild notochord deformities. The coronal and sagittal planes are reconstructed from μCT images of the fish in the upper panels, each showing a gross appearance. The red arrows indicate vertebral malformation. (F) Diagram of zebrafish growth stages. Continuous observation of one <i>GATA2</i>-<i>1b</i>:<i>lbx1b</i>-injected larva with a displaced dorsal melanophore stripe, but without notochord deformation, from 6 to 90 dpf. The red and green dotted lines indicate the dorsal middle lines and the right upper boundary of the lateral stripe, respectively. Progressive scoliosis with rotation of the longitudinal axis of the body was observed after 55 dpf. E, embryo; L, larva; J, juvenile; A, adult. (G) μCT analysis of the three-dimensional structure of the spine of the same zebrafish in (F) showing scoliosis. Cobb’s angle was measured in the coronal plane. Scale bars in (B): 200 μm; (C): 1 mm or 100 μm; (D): 500 μm; and (F): 1 mm.</p