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Integrin beta4 regulation of PTHrP underlies its contribution to mammary gland development

Wed, 12/23/2015 - 9:31am

The integrin alpha6beta4 (referred to as beta4) is expressed in epithelial cells where it functions as a laminin receptor. Although in vitro studies have implicated beta4 in the biology of mammary epithelial cells, its contribution to mammary gland development has not been settled. To address this problem, we generated and analyzed itgb4(flox/flox)MMTV-Cre(-) and itgb4(flox/flox)MMTV-Cre(+) mice. The salient features of embryonic mammary tissue from itgb4(flox/flox)MMTV-Cre(+) mice were significantly smaller mammary buds and increased apoptosis in the surrounding mesenchyme. Also, compared to control glands, the itgb4-deleted mammary buds lacked expression of the progenitor cell marker CK14 and they were unable to generate mammary glands upon transplantation into cleared fat pads of recipient mice. Analysis of mammary glands at puberty and during pregnancy revealed that itgb4-diminished mammary tissue was unable to elongate and undergo branching morphogenesis. Micro-dissection of epithelial cells in the mammary bud and of the surrounding mesenchyme revealed that loss of beta4 resulted in a significant decrease in the expression of parathyroid hormone related protein (PTHrP) in epithelial cells and of target genes of the PTHrP receptor in mesenchymal cells. Given that the phenotype of the itgb4-deleted mammary tissue mimicked that of the PTHrP knockout, we hypothesized that beta4 contributes to mammary gland development by sustaining PTHrP expression and enabling PTHrP signaling. Indeed, the inability of itgb4-deleted mammary buds to elongate was rescued by exogenous PTHrP. These data implicate a critical role for the beta4 integrin in mammary gland development and provide a mechanism for this role.

SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef

Wed, 12/23/2015 - 9:31am

HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

Yeast as Models of Mitotic Fidelity

Wed, 12/23/2015 - 9:31am

Chromosome missegregation leads to aneuploidy which is defined as the cellular state of having a chromosome count that is not an exact multiple of the haploid number. Aneuploidy is associated with human diseases including mental retardation, neurodegenerative diseases and cancer. In addition, aneuploidy is the major cause of spontaneous abortions and its occurrence increases with aging. Therefore, it is important to understand the molecular mechanisms by which cells respond and adapt to aneuploidy. Saccharomyces cerevisiae has proven to be a good model to study the effects aneuploidy elicits on cellular homeostasis and physiology. This chapter focuses on the current understanding of how the yeast S. cerevisiae responds to the acquisition of extra chromosomes and highlights how studies in aneuploid yeasts provide insights onto the effects of aneuploidy in human cells.

Want reprogramming? Cut back on the chromatin assembly!

Wed, 12/23/2015 - 9:31am

Totipotency, the ability of early embryonic cells to generate a complete adult organism as well as extraembryonic tissue, is a fleeting property found only in very early embryonic cells. A breakthrough study now shows that inhibition of DNA replication–linked nucleosome assembly causes embryonic stem cells to resemble totipotent cells. Notably, inhibition of chromatin assembly stimulates reprogramming during somatic-cell nuclear transfer experiments.

Homozygous knockout of the piezo1 gene in the zebrafish is not associated with anemia

Wed, 12/23/2015 - 9:31am

We have now examined the erythroid phenotype in this zebrafish strain carrying a ZFN genomic knockout of piezo1. Genotyping was performed as previously described. In contrast to the anemic phenotype observed in zebrafish subjected to morpholino knockdown of piezo, the genomic ZFN knockout of piezo1 did not segregate either with anemia in the 3-dpf embryo or with dysmorphic erythrocyte morphology in the adult fish.

Prenatal Maternal Physical Activity and Stem Cells in Umbilical Cord Blood

Wed, 12/23/2015 - 9:31am

PURPOSE: Early life processes, through influence on fetal stem cells, affect postnatal and adult health outcomes. This study examines effects of physical activity before and during pregnancy on stem cell counts in umbilical cord blood.

METHODS: We isolated mononuclear cells from umbilical cord blood samples from 373 singleton full-term pregnancies and quantified hematopoietic (CD34, CD34CD38, CD34c-kit), endothelial (CD34CD133, CD34CD133VEGFR2, CD34VEGFR2, and CD133VEGFR2), and putative breast (EpCAM, EpCAMCD49f, EpCAMCD49fCD117, CD49fCD24, CD24CD29, and CD24CD29CD49f) stem/progenitor cell subpopulations by flow cytometry. Information on physical activities before and during pregnancy was obtained from questionnaire. Weekly energy expenditure was estimated based on the metabolic equivalent task (MET) values.

RESULTS: Pre-pregnancy vigorous exercise was associated positively with levels of the endothelial CD34CD133, CD34CD133VEGFR2, CD34VEGFR2, and CD133VEGFR2 progenitor cell populations (p=0.02, 0.01, 0.001, and 0.003, respectively); the positive associations were observed in samples from the first births and those from the second or later births. Pre-pregnancy moderate and light exercise and light exercise during the first trimester were not significantly associated with any stem/progenitor cell population. Light exercise during the second trimester was positively associated with CD34VEGFR2 endothelial progenitor cells (p=0.03). In addition, levels of the EpCAMCD49f and CD49fCD24 breast stem cells were significantly lower among pregnant women who engaged in vigorous or moderate exercise during pregnancy (p=0.05 and 0.02, respectively).

CONCLUSION: Vigorous exercise before pregnancy increases endothelial progenitor cell numbers in umbilical cord blood and thus could potentially enhance the endothelial function and improve cardiovascular fitness in the offspring. Findings of a lower level of putative breast stem cell sub-populations could have implication on exercise and breast cancer prevention. Prenatal effects of exercise on fetal stem cells warrant further studies.

Grabbing the genome by the NADs

Wed, 12/23/2015 - 9:31am

The regions of the genome that interact frequently with the nucleolus have been termed nucleolar-associated domains (NADs). Deep sequencing and DNA-fluorescence in situ hybridization (FISH) experiments have revealed that these domains are enriched for repetitive elements, regions of the inactive X chromosome (Xi), and several RNA polymerase III-transcribed genes. NADs are often marked by chromatin modifications characteristic of heterochromatin, including H3K27me3, H3K9me3, and H4K20me3, and artificial targeting of genes to this area is correlated with reduced expression. It has therefore been hypothesized that NAD localization to the nucleolar periphery contributes to the establishment and/or maintenance of heterochromatic silencing. Recently published studies from several multicellular eukaryotes have begun to reveal the trans-acting factors involved in NAD localization, including the insulator protein CCCTC-binding factor (CTCF), chromatin assembly factor (CAF)-1 subunit p150, several nucleolar proteins, and two long non-coding RNAs (lncRNAs). The mechanisms by which these factors coordinate with one another in regulating NAD localization and/or silencing are still unknown. This review will summarize recently published studies, discuss where additional research is required, and speculate about the mechanistic and functional implications of genome organization around the nucleolus.

A platform for reverse genetics in endothelial cells

Wed, 12/23/2015 - 9:31am

The recent development of programmable nucleases has the potential to revolutionize biological sciences. In particular, the Cas9 nuclease, which functions as a component of the clustered regularly interspaced short palindromic repeats (CRISPR) system in bacteria, has proven to be a highly efficient tool for genome editing in a wide range of model organisms, including mouse, zebrafish,Drosophila, and Caenorhabditis elegans. Application of Cas9 also allows straightforward genetic manipulations in cultured cells and is efficient enough to perform genome-wide screens in cell lines. However, applying genome editing tools in this manner in vascular biology is challenging because of the widespread use of primary cell cultures, which have a limited lifespan and are difficult to use for clonal analysis. Fortunately, studies by Abrahimi et al in this issue describe several solutions that facilitate the application of Cas9 in cultured endothelial cells. Together, these technical advances provide a valuable platform to enable straightforward and robust reverse genetic analysis in endothelial cells.

Using C. elegans for aging research

Wed, 12/23/2015 - 9:31am

Over a century ago, the zoologist Emile Maupas first identified the nematode, Rhabditis elegans, in the soil in Algiers. Subsequent work and phylogenic studies renamed the species Caenorhabditis elegans or more commonly referred to as C. elegans; (Caeno meaning recent; rhabditis meaning rod; elegans meaning nice). However, it was not until 1963, when Sydney Brenner, already successful from his work on DNA, RNA, and the genetic code, suggested the future of biological research lay in model organisms. Brenner believed that biological research required a model system that could grow in vast quantities in the lab, were cheap to maintain and had a simple body plan, and he chose the nematode C. elegans to fulfill such a role. Since that time, C. elegans has emerged as one of the premiere model systems for aging research. This paper reviews some initial identification of mutants with altered lifespan with a focus on genetics and then discusses advantages and disadvantages for using C. elegans as a model system to understand human aging. This review focuses on molecular genetics aspects of this model organism.

IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions

Wed, 12/23/2015 - 9:31am

The endoplasmic reticulum (ER) has emerged as a critical regulator of cell survival. IRE1 is a transmembrane protein with kinase and RNase activities that is localized to the ER and that promotes resistance to ER stress. We showed a mechanism by which IRE1 conferred protection against ER stress-mediated cell death. IRE1 signaling prevented ER membrane permeabilization mediated by Bax and Bak and cell death in cells experiencing ER stress. Suppression of IRE1 signaling triggered by its kinase activity led to the accumulation of the BH3 domain-containing protein Bnip3, which in turn triggered the oligomerization of Bax and Bak in the ER membrane and ER membrane permeabilization. Consequently, in response to ER stress, cells deficient in IRE1 were susceptible to leakage of ER contents, which was associated with the accumulation of calcium in mitochondria, oxidative stress in the cytosol, and ultimately cell death. Our results reveal a role for IRE1 in preventing a cell death-initializing step that emanates from the ER and provide a potential target for treating diseases characterized by ER stress, including diabetes and Wolfram syndrome.

Global Promotion of Alternative Internal Exon Usage by mRNA 3' End Formation Factors

Wed, 12/23/2015 - 9:30am

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

Family history and risk of pregnancy-associated breast cancer (PABC)

Wed, 12/23/2015 - 9:30am

The risk of breast cancer is at least two-fold increased in young women with a family history of breast cancer. Pregnancy has a dual effect on breast cancer risk; a short-term increase followed by a long-term protection. We investigated if the risk of breast cancer during and within 10 years following pregnancy is affected by a family history of breast cancer. We followed a cohort of women aged 15-44 years between 1963 and 2009 identified in Swedish population-based registers. Family history was defined as having a mother or sister with breast cancer. We estimated incidence rate ratios of breast cancer during pregnancy and time intervals up to 10 years post-delivery, with a focus on pregnancy-associated breast cancer (PABC), defined as breast cancer during pregnancy or within 2 years post-delivery. In 3,452,506 women, there were 15,548 cases of breast cancer (1208 were PABC). Compared to nulliparous women, the risk of breast cancer was decreased during pregnancy, similar during first year and increased during second year post-delivery. The pattern was similar in women with or without family history of breast cancer. A peak in risk was observed 5-6 years following the first birth regardless of family history. After a second birth, this peak was only present in women with a family history. Our results indicate that women with a family history of breast cancer do not have a different breast cancer risk during and within 10 years following pregnancy compared to women without a family history.

Research resource: Monitoring endoplasmic reticulum membrane integrity in beta-cells at the single-cell level

Wed, 12/23/2015 - 9:30am

Endoplasmic reticulum (ER) membrane integrity is an emerging target for human chronic diseases associated with ER stress. Despite the underlying importance of compromised ER membrane integrity in disease states, the entire process leading to ER membrane permeabilization and cell death is still not clear due to technical limitations. Here we describe a novel method for monitoring ER membrane integrity at the single-cell level in real time. Using a beta-cell line expressing ER-targeted redox sensitive green fluorescent protein, we could identify a beta-cell population undergoing ER membrane permeabilization induced by palmitate and could monitor cell fate and ER stress of these cells at the single-cell level. Our method could be used to develop a novel therapeutic modality targeting the ER membrane for ER-associated disorders, including beta-cell death in diabetes, neurodegeneration, and Wolfram syndrome.

TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein

Wed, 12/23/2015 - 9:30am

The TRIM37 (also known as MUL) gene is located in the 17q23 chromosomal region, which is amplified in up to approximately 40% of breast cancers. TRIM37 contains a RING finger domain, a hallmark of E3 ubiquitin ligases, but its protein substrate(s) is unknown. Here we report that TRIM37 mono-ubiquitinates histone H2A, a chromatin modification associated with transcriptional repression. We find that in human breast cancer cell lines containing amplified 17q23, TRIM37 is upregulated and, reciprocally, the major H2A ubiquitin ligase RNF2 (also known as RING1B) is downregulated. Genome-wide chromatin immunoprecipitation (ChIP)-chip experiments in 17q23-amplified breast cancer cells identified many genes, including multiple tumour suppressors, whose promoters were bound by TRIM37 and enriched for ubiquitinated H2A. However, unlike RNF2, which is a subunit of polycomb repressive complex 1 (PRC1), we find that TRIM37 associates with polycomb repressive complex 2 (PRC2). TRIM37, PRC2 and PRC1 are co-bound to specific target genes, resulting in their transcriptional silencing. RNA-interference-mediated knockdown of TRIM37 results in loss of ubiquitinated H2A, dissociation of PRC1 and PRC2 from target promoters, and transcriptional reactivation of silenced genes. Knockdown of TRIM37 in human breast cancer cells containing amplified 17q23 substantially decreases tumour growth in mouse xenografts. Conversely, ectopic expression of TRIM37 renders non-transformed cells tumorigenic. Collectively, our results reveal TRIM37 as an oncogenic H2A ubiquitin ligase that is overexpressed in a subset of breast cancers and promotes transformation by facilitating silencing of tumour suppressors and other genes.

Autophagy in Cell Life and Cell Death

Wed, 12/23/2015 - 9:30am

Macroautophagy (hereafter referred to as autophagy) is a process used by the cell to deliver cytoplasmic components to the lysosome for degradation. Autophagy is most often associated with cell survival, as it provides cells with molecular building blocks during periods of nutrient deprivation and also aids in the elimination of damaged organelles and protein aggregates. However, autophagy has also been implicated in cell death. Here, we review what is known about autophagy, its regulation, its role both in cell life and cell death, and what is known about autophagic cell death in vivo.

The Sound of Silence: Signaling by Apoptotic Cells

Wed, 12/23/2015 - 9:30am

Apoptosis is a carefully choreographed process of cellular self-destruction in the absence of inflammation. During the death process, apoptotic cells actively communicate with their environment, signaling to both their immediate neighbors as well as distant sentinels. Some of these signals direct the anti-inflammatory immune response, instructing specific subsets of phagocytes to participate in the limited and careful clearance of dying cellular debris. These immunomodulatory signals can also regulate the activation state of the engulfing phagocytes. Other signals derived from apoptotic cells contribute to tissue growth control with the common goal of maintaining tissue integrity. Derangements in these growth control signals during prolonged apoptosis can lead to excessive cell loss or proliferation. Here, we highlight some of the most intriguing signals produced by apoptotic cells during the course of normal development as well as during physiological disturbances such as atherosclerosis and cancer.

Global Promotion of Alternative Internal Exon Usage by mRNA 3' End Formation Factors

Wed, 12/23/2015 - 9:30am

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

The CREB Coactivator CRTC2 Is a Lymphoma Tumor Suppressor that Preserves Genome Integrity through Transcription of DNA Mismatch Repair Genes

Wed, 12/23/2015 - 9:30am

The CREB-regulated transcription coactivator CRTC2 stimulates CREB target gene expression and has a well-established role in modulating glucose and lipid metabolism. Here, we find, unexpectedly, that loss of CRTC2, as well as CREB1 and its coactivator CREB-binding protein (CBP), results in a deficiency in DNA mismatch repair (MMR) and a resultant increased mutation frequency. We show that CRTC2, CREB1, and CBP are transcriptional activators of well-established MMR genes, including EXO1, MSH6, PMS1, and POLD2. Mining of expression profiling databases and analysis of patient samples reveal that CRTC2 and its target MMR genes are downregulated in specific T cell lymphoma subtypes, which are microsatellite unstable. The levels of acetylated histone H3 on the CRTC2 promoter are significantly reduced in lymphoma in comparison to normal tissue, explaining the decreased CRTC2 expression. Our results establish a role for CRTC2 as a lymphoma tumor suppressor gene that preserves genome integrity by stimulating transcription of MMR genes.

Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages

Wed, 12/23/2015 - 9:30am

During development, progenitor cells with binary potential give rise to daughter cells that have distinct functions. Heritable epigenetic mechanisms then lock in gene-expression programs that define lineage identity. Regulation of the gene encoding the T cell-specific coreceptor CD4 in helper and cytotoxic T cells exemplifies this process, with enhancer- and silencer-regulated establishment of epigenetic memory for stable gene expression and repression, respectively. Using a genetic screen, we identified the DNA-methylation machinery as essential for maintaining silencing of Cd4 in the cytotoxic lineage. Furthermore, we found a requirement for the proximal enhancer in mediating the removal of DNA-methylation marks from Cd4, which allowed stable expression of Cd4 in helper T cells. Our findings suggest that stage-specific methylation and demethylation events in Cd4 regulate its heritable expression in response to the distinct signals that dictate lineage 'choice' during T cell development.

F-box protein FBXO31 directs degradation of MDM2 to facilitate p53-mediated growth arrest following genotoxic stress

Wed, 12/23/2015 - 9:30am

The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage.