Human Gene Family Research Articles

Novel and Unexpected Functions of SGLTs.

It has been 30 years since the intestinal sodium glucose cotransporter SGLT1 was cloned, and, in the intervening years, there have been many advances that have influenced physiology and medicine. Among the first was that SGLT1 is the founding member of the human gene family SLC5, containing 11 diverse transporters and a glucose sensor. Equally surprising was that SGLTs are members of a structural family of cotransporters and exchangers in different gene families. This led to the conclusion that these proteins operate by a mechanism where transport involves the opening and closing of external and internal gates. The mechanism is shared by a wide variety of transporters in different structural families, e.g., the human facilitated glucose transporters (SLC2) in the huge major facilitator superfamily (MFS). Not surprising is the finding that mutations in Sglt genes cause the rare diseases glucose-galactose-malabsorption (GGM) and familial renal glucosuria (FRG). However, it was not envisaged that SGLT inhibitors would be used to treat diabetes mellitus, and these drugs may be able to treat cancer. Finally, in 2017, we have just learned that SGLT1 may be required to resist infection and to avoid recurrent pregnancy loss.

Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies.

The rhesus macaque is a critically important animal model in biomedical research, most recently playing a key role in the development of vaccines against human immunodeficiency virus-1. Nevertheless, the immunoglobulin (Ig) loci of macaques are as yet incompletely determined and our understanding of differences between human and macaque humoral immunity remains deficient. We completed a high-coverage, high-quality whole genome sequencing and assembly project with a single rhesus macaque of Indian origin, and partial genome assemblies using genomic molecular targeting of the Ig loci in nine other rhesus macaques of Indian origin. These data indicate that the macaque Ig loci are substantially more diverse than those in humans, including greater sequence diversity and copy-number variation between individuals. It appears likely that such copy-number variation even occurs between allelic loci within individuals. Different Ig gene families in the macaque show distinct relationships to the corresponding human gene families and appear to evolve under different mechanisms. These results raise intriguing questions about the evolution of antigen receptors in primates but also have important practical implications for the design and interpretation of biomedical studies.

Human TLR gene family members are differentially expressed in patients with urothelial carcinoma of the bladder

PurposeToll-like receptors (TLRs) have an important role in the activation of both innate and adaptive immunity in response to pathogens and endogenous danger signals from damaged or dying cells. The aim of this study was to determine the relationship between urothelial carcinoma (UC) and TLR expression. Basic proceduresReal-time polymerase chain reaction evaluation was made of the messenger RNA expression of TLRs 1–10 in 24 UC samples and 46 nontumoral bladder tissue samples. The levels of proinflammatory cytokines (IL-1β, IL-6, and IL-8) in the urine samples were also determined with enzyme-linked immunosorbent assay. Main findingsTLR2–7 and TLR10 expressions were significantly higher in UC than in the control group (P<0.05 for all comparisons). No concordance was found between matched tumor tissue and urine samples in terms of TLR expression. IL-1β, IL-6, and IL-8 levels were significantly higher in urine specimens of patients with UC (P = 0.033, P = 0.001, and P = 0.008, respectively). Principal conclusionsThe results of this study demonstrated that the TLR gene expression profiles reflect the heterogeneity within UC. These results might also prompt further investigation to better understand the role of the TLR gene family expression in the tumor progression of UC.

Host genes regulate transcription of sperm-introduced hepatitis B virus genes in embryo

Hepatitis B virus (HBV) can invade the male germline, and sperm-introduced HBV genes could be transcribed in embryo. This study was to explore whether viral gene transcription is regulated by host genes. Embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the HBV genome. Total RNA extracted from test and control embryos were subjected to SMART-PCR, SSH, microarray hybridization, sequencing and BLAST analysis. Twenty-nine sequences showing significant identity to five human gene families were identified, with CSH2, EIF4G2, PCBD2, PSG4 and TTN selected to represent target genes. Using qRT-PCR, when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNAi, transcriptional levels of HBV s and x genes decreased (or increased). This is the first report that host genes participate in regulation of sperm-introduced HBV gene transcription in embryo, which is critical to prevent negative impact of HBV infection on early embryonic development.

Abstract 854: Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer

Abstract Background: Kallikrein-related peptidase 6 (KLK6) belongs to the family of human tissue kallikrein genes, majority of which are shown to be differentially expressed in cancers. Clinical studies have demonstrated that upregulation of KLK6 in primary colorectal tumors and lymph nodes correlates with serosal invasion, liver metastasis and indicative of poor prognosis in patients. It has been reported that KLK6 protein is involved in regulation of the epithelial-mesenchymal transition (EMT) program in an organ-specific context. The aim in this study was to investigate contribution of KLK6 enzyme in the EMT during neoplastic transformation in the colon. Results: We expressed enzymatically active or inactive KLK6, using pcDNA3.1(+)preproKLK6 and pcDNA3.1(+)preproKLK6 Ser197Ala mutant plasmids, in Caco-2 colon cancer cell line, which has been characterized before as a very low KLK6 expresser with an undetectable secreted KLK6. Stable isogenic clones were selected and further evaluated for their ability to migrate and invade using in vitro assays and to metastasize in vivo using SCID orthotopic mouse model. We found no effect of KLK6 enzyme activity on migration of Caco-2 cells, expressing the empty vector (Caco-2 mock), and Caco-2 cells, expressing an enzymatically active KLK6 (Caco-2 KLK6 wt) or inactive KLK6 (Caco-2 KLK6 mut). But Caco-2 KLK6 wt cells demonstrated the invasive phenotype in Matrigel invasion assays (p&amp;lt;0.001, compared to Caco-2 mock and Caco2 KLK6 mut cells). The Caco-2 mock and Caco-2 KLK6 mut cells, injected in SCID mice orthotopically, developed primary colon tumors but no metastatic lesions were identified. In contrast, Caco-2 KLK6 wt cells formed primary colon tumors and metastasized locally, although they failed to form the distant metastasis (lung and mesentery). Animals, growing the Caco-2 KLK6 wt tumors, displayed a significant decrease in their survival rates, compared to other groups (p=0.02). In Caco-2 KLK6 wt cells TGF-β protein expression and secretion was induced, which resulted in activation of TGF-β-SMAD2/3 signaling pathway. This phenotype was associated with the elevated expression of known regulator of the EMT, zinc-finger protein Snail. In addition, the expression of a high-mobility group AT-hook 2 (HMGA2) protein was induced in Caco-2 KLK6 wt cells. The HMGA2 expression is implicated in the EMT program, acting through the TGF-β signaling pathway and is associated with a poor survival in colorectal cancer. Conclusion. These findings demonstrate that KLK6 enzyme activity is required for colon cancer progression via induction of the EMT program. We identified the TGF-β- signaling pathway as a mechanism driving the EMT in colon cancer cells expressing KLK6 enzyme. Citation Format: Hwudaurw Chen, Earlphia Sells, Haiyan Cui, Ritu Pandey, George Pampalakis, Georgia Sotiropoulou, Thomas Doetschman, Natalia A. Ignatenko. Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 854. doi:10.1158/1538-7445.AM2017-854

Role of the Vanins-Myeloperoxidase Axis in Colorectal Carcinogenesis.

The presence of chronic inflammation in the colonic mucosa leads to an increased risk of cancer. Among proteins involved in the regulation of mucosal inflammation and that may contribute both to structural damage of the intestinal mucosa and to intestinal carcinogenesis, there are myeloperoxidase (MPO) and vanins. The infiltration of colonic mucosa by neutrophils may promote carcinogenesis through MPO, a key enzyme contained in the lysosomes of neutrophils that regulates local inflammation and the generation of reactive oxygen species (ROS) and mutagenic species. The human vanin gene family consists of three genes: vanin-1, vanin-2 and vanin-3. All vanin molecules are pantetheinases, that hydrolyze pantetheine into pantothenic acid (vitamin B5), and cysteamine, a sulfhydryl compound. Vanin-1 loss confers an increased resistance to stress and acute intestinal inflammation, while vanin-2 regulates adhesion and transmigration of activated neutrophils. The metabolic product of these enzymes has a prominent role in the inflammation processes by affecting glutathione levels, inducing ulcers through a reduction in mucosal blood flow and oxygenation, decreasing local defense mechanisms, and in carcinogenesis by damaging DNA and regulating pathways involved in cell apoptosis, metabolism and growth, as Nrf2 and HIF-1α.

Using Engineered Recombinant Tubulin Sotypes to Study VDAC Regulation

Highly variable anionic C-terminal tails (CTTs) of tubulin had been implicated in regulation of permeability of the mitochondrial voltage-dependent anion channel (VDAC). Tubulin, a structural protein of microtubules, is a heterodimer comprising α/β subunits. In cells tubulin possesses significant variability due to the expression of several tubulin genes. Seven α- and eight β-tubulin isoforms in the human tubulin gene family vary mostly in the length and primary sequence of the CTTs. This inherent non-homogeneity of the endogenous tubulin CTTs hindered the analysis of their interaction with VDAC reconstituted into planar lipid bilayers. A recent study (Sheldon et al., 2015) with peptide-protein chimeras evaluated the frequency of the VDAC blockage events to demonstrate that the on-rate is highly dependent on the tail chemical composition. In that study CTT-like peptides were grafted to albumin, a protein that does not bind to the membrane surface, though it is now conclusively established that membrane binding of the tubulin body is an essential step in tubulin/VDAC interaction. Here we took an advantage of recently generated recombinant yeast α/β-tubulin constructs with CTTs from various human tubulin isotypes (Sirajuddin et al., 2014). The effects of each of the engineered constructs on the reconstituted VDAC were compared to those of w.t. yeast tubulin and recombinant tubulin without CTTs. Single-molecule studies of the tubulin-VDAC interaction showed that length and total anionic charge of the individual CTTs correlate strongly with the average duration of the blockage. In particular, at 30-mV applied voltage the characteristic time of VDAC blockage by the longer and more highly charged β3-CTT is up to hundredfold greater than that observed for the α1-CTT, with the apparent gating charge doubled. These results suggest novel implications in VDAC regulation by the diversity of tubulin isotypes found in vertebrates.

MYC and RAF: Key Effectors in Cellular Signaling and Major Drivers in Human Cancer.

The prototypes of the human MYC and RAF gene families are orthologs of animal proto-oncogenes that were originally identified as transduced alleles in the genomes of highly oncogenic retroviruses. MYC and RAF genes are now established as key regulatory elements in normal cellular physiology, but also as major cancer driver genes. Although the predominantly nuclear MYC proteins and the cytoplasmic RAF proteins have different biochemical functions, they are functionally linked in pivotal signaling cascades and circuits. The MYC protein is a transcription factor and together with its dimerization partner MAX holds a central position in a regulatory network of bHLH-LZ proteins. MYC regulates transcription conducted by all RNA polymerases and controls virtually the entire transcriptome. Fundamental cellular processes including distinct catabolic and anabolic branches of metabolism, cell cycle regulation, cell growth and proliferation, differentiation, stem cell regulation, and apoptosis are under MYC control. Deregulation of MYC expression by rearrangement or amplification of the MYC locus or by defects in kinase-mediated upstream signaling, accompanied by loss of apoptotic checkpoints, leads to tumorigenesis and is a hallmark of most human cancers. The critically controlled serine/threonine RAF kinases are central nodes of the cytoplasmic MAPK signaling cascade transducing converted extracellular signals to the nucleus for reshaping transcription factor controlled gene expression profiles. Specific mutations of RAF kinases, such as the prevalent BRAF(V600E) mutation in melanoma, or defects in upstream signaling or feedback loops cause decoupled kinase activities which lead to tumorigenesis. Different strategies for pharmacological interference with MYC- or RAF-induced tumorigenesis are being developed and several RAF kinase inhibitors are already in clinical use.

Monogenic and Monoallelic Expression of Odorant Receptors

Odorant receptors (ORs) belong to a large gene family of rhodopsin-like G protein-coupled receptors (GPCRs). The mouse OR gene family is composed of ∼1000 OR genes, and the human OR gene family is composed of ∼400 OR genes. The OR genes are spread throughout the genome, and can be found in clusters or as solitary genes in almost all chromosomes. These chemosensory GPCRs are expressed in highly specialized cells, the olfactory sensory neurons of the nose. Each one of these neurons expresses a single OR gene out of the complete repertoire of genes. In addition, only one of the two homologous alleles of the chosen OR gene, the maternal or the paternal, is expressed per neuron. Here we review recent findings that help to elucidate the mechanisms underlying monogenic and monoallelic expression of OR genes.

Unfavorable neuroblastoma prognostic factor NLRR2 inhibits cell differentiation by transcriptional induction through JNK pathway.

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)‐mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA‐induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c‐Jun, a member of the activator protein‐1 (AP‐1) family in NB cells. Moreover, the expressions of NLRR2 and c‐Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c‐Jun reduced NLRR2 expression. We then searched AP‐1 binding consensus in the NLRR2 promoter region and confirmed c‐Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.

Unveiling alterative splice diversity from human oligodendrocyte proteome data

Oligodendrocytes produce and maintain the myelin sheath of axons in the central nervous system. Because misassembled myelin sheaths have been associated with brain disorders such as multiple sclerosis and schizophrenia, recent advances have been made towards the description of the oligodendrocyte proteome. The identification of splice variants represented in the proteome is as important as determining the level of oligodendrocyte-associated proteins. Here, we used an oligodendrocyte proteome dataset deposited in ProteomeXchange to search against a customized protein sequence file containing computationally predicted splice variants. Our approach resulted in the identification of 39 splice variants, including one variant from the GTPase KRAS gene and another from the human glutaminase gene family. We also detected the mRNA expression of five selected splice variants and demonstrated that a fraction of these have their canonical proteins participating in direct protein-protein interactions. In conclusion, we believe our findings contribute to the molecular characterization of oligodendrocytes and may encourage other research groups working with central nervous system disorders to investigate the biological significance of these splice variants. The splice variants identified in this study may encode proteins that could be targeted in novel treatment strategies and diagnostic methods. SignificanceSeveral disorders of the central nervous system (CNS) are associated with misassembled myelin sheaths, which are produced and maintained by oligodendrocytes (OL). Recently, the OL proteome has been explored to identify key proteins and molecular functions associated with CNS disorders. We developed an innovative approach to select, with a higher level of confidence, a relevant list of splice variants from a proteome dataset and detected the mRNA expression of five selected variants: EEF1D, KRAS, MFF, SDR39U1, and SUGT1. We also described splice variants extracted from OL proteome data. Among the splice variants identified, some are from genes previously linked to CNS and related disorders. Our findings may contribute to oligodendrocyte characterization and encourage other research groups to investigate the biological role of splice variants and to improve current treatments and diagnostic methods for CNS disorders.

Molecular adaption of alcohol metabolism to agriculture in East Asia

Abstract Human Alcohol metabolic gene families ADH and ALDH mainly comprise ten genes, expressing in different organs and tissues. Two of the variants, ADH1B*47His and ALDH2*504Lys reach very high frequency in East Asia, while are almost absent in the rest of the world, which is believed to be results of positive selection related to the development of agriculture in Neolithic time. In addition to the ADH alcohol metabolic pathway, a microsomal ethanol oxidizing system involving the gene CYP2E1 has also been identified. The study on the micro-evolution of alcohol metabolic genes will help us understand how human adapted to the artificial environment of agriculture. We collected 1211 samples from 44 worldwide populations including 19 representative populations in East Asia, combined with 2504 samples of 26 populations from 1000 Genome project. We scanned all the 23 missense mutations or pathogenic SNPs in ADH and CYP2E1 pathways. Then we examined the selection signature on the genetic polymorphism in East Asia and estimated the allele ages. Our analyses revealed that the long-term farming ethnic groups in East Asia, such as Han, differed from the nomadic populations in the pattern of alcohol-related genetic polymorphism. This divergence was mainly attributed to the 6 closely related functional SNPs rs1229984 ( ADH1B ), rs671 ( ALDH2 ), rs8187929 ( ALDH1A1 ), rs2228093 ( ALDH1B1 ), rs3813867 ( CYP2E1 ), and rs2031920 ( CYP2E1 ). The derived core haplotypes of the new detected SNPs showed moderate to strong selection signals and the estimated allele ages coincided with the Neolithic time. The driving force tended to be the emergence and expansion of agriculture in East Asia.

Human growth gene family chromosomal architecture is affected by maternal obesity, gestational diabetes and insulin treatment

Human placental chorionic somatomammotropin (CS) ‐A and B, and growth hormone variant (GH‐V) are placental members of the human (h) growth hormone (GH) gene family. These genes are located at a single locus on chromosome 17 in tandem with the pituitary hGH gene (GH‐N). CS and GH‐V stimulate pancreatic β‐cells in the response to ‘normal’ pregnancy induced insulin resistance. Maternal obesity exacerbates this ‘resistance’ and decreases CS/GH‐V gene expression, increasing the risk for gestational diabetes (GDM). Looping together of hGH‐N promoter and remote (14.5–32 kilobases) upstream hGH/CS locus control region (LCR) sequences is essential for efficient gene expression in vivo. Recently, hGH‐N chromatin looping and gene expression were shown to be negatively affected by excess caloric intake. Thus, we have compared the effect of maternal obesity (BMI &gt;35 kg/m2) without or with the further complication of GDM and insulin treatment on the chromatin structure and expression of the hCS/GH‐V gene locus in pregnancy. Human CS‐A RNA was decreased with maternal obesity but increased to levels detected in placentas from obese women with insulin treatment. Chromatin conformation capture (3C) assay was used to assess the long‐range interaction between the remote LCR sequences that include three nuclease hypersensitive sites (HS III–V) in human placental syncytiotrophoblasts, with and more proximal regulatory sequences linked to CS/GH‐V gene expression. Recruitment of RNA polymerase II at the promoter regions was also assessed as an indication of transcriptional status by chromatin immunoprecipitation assay. Physical interactions between remote LCR and local regulatory regions of the CS genes, suggesting looping of intervening sequences, were detected in lean woman, but were disrupted by maternal obesity. The same disrupted chromosomal conformation was not seen in obese diabetic pregnant women treated with insulin. However, although evidence of chromatin looping involving LCR and local regulatory sequences was apparent, the pattern was distinct when compared to lean placental samples. Maternal obesity was associated with significant decreases in RNA polymerase II occupancy at the promoter regions. Insulin treatment in obese women with a diabetic condition, however, significantly muted the decrease in RNA polymerase II occupancy at the CS/GH‐V promoter regions, presumably reflecting the looping of regulatory regions that was observed. Thus, insulin treatment of obese women with GDM results in overall CS/GH‐V RNA levels comparable to those seen in lean women. Recovery of the CS transcript levels, however, does not correlate with the preservation of the three dimensional chromosomal structure of the placental GH gene locus as defined in normal pregnancies. These data suggest a vulnerability of the hGH/CS gene locus chromatin structure and genes to excess caloric intake in pregnancy but also some plasticity in terms of response.Support or Funding InformationFunded by the Canadian Institutes of Health Research

Distinct lymphocyte antigens 6 (Ly6) family members Ly6D, Ly6E, Ly6K and Ly6H drive tumorigenesis and clinical outcome.

Stem cell antigen-1 (Sca-1) is used to isolate and characterize tumor initiating cell populations from tumors of various murine models [1]. Sca-1 induced disruption of TGF-β signaling is required in vivo tumorigenesis in breast cancer models [2, 3-5]. The role of human Ly6 gene family is only beginning to be appreciated in recent literature [6-9]. To study the significance of Ly6 gene family members, we have visualized one hundred thirty gene expression omnibus (GEO) dataset using Oncomine (Invitrogen) and Georgetown Database of Cancer (G-DOC). This analysis showed that four different members Ly6D, Ly6E, Ly6H or Ly6K have increased gene expressed in bladder, brain and CNS, breast, colorectal, cervical, ovarian, lung, head and neck, pancreatic and prostate cancer than their normal counter part tissues. Increased expression of Ly6D, Ly6E, Ly6H or Ly6K was observed in sub-set of cancer type. The increased expression of Ly6D, Ly6E, Ly6H and Ly6K was found to be associated with poor outcome in ovarian, colorectal, gastric, breast, lung, bladder or brain and CNS as observed by KM plotter and PROGgeneV2 platform. The remarkable findings of increased expression of Ly6 family members and its positive correlation with poor outcome on patient survival in multiple cancer type indicate that Ly6 family members Ly6D, Ly6E, Ly6K and Ly6H will be an important targets in clinical practice as marker of poor prognosis and for developing novel therapeutics in multiple cancer type.

In silico analysis of histone H3 gene expression during human brain development.

Precise regulation of chromatin structure is essential for proper development of higher eukaryotes, and methylation of histone H3 at lysine-27 (H3K27) by the Polycomb Repressive Complex 2 (PRC2) component EZH2 has emerged as an important and conserved mechanism to ensure silencing of developmentally regulated genes. Recurrent mutations within the histone H3 genes H3F3A and HIST1H3B that convert K27 to methionine (H3K27M) and disrupt the global H3K27 methylation landscape and PRC2-dependent silencing, have recently been identified in pediatric high-grade gliomas including Diffuse Intrinsic Pontine Glioma (DIPG) and Glioblastoma multiforme (GBM; Type IV glioma). These findings have generated renewed interest in the dynamics of histone genes and their expression, which have been difficult to study due to redundancy and high sequence homology within the H3 gene family. In this in silico study, we re-evaluated genomic organization of the human H3 gene family and expression of these genes in the human brain, utilizing public RNA-based sequence datasets for the human genome and brain development. We identified transcriptional activity from at least 17 protein-encoding H3 genes in the developing brain, comprising at least 14 canonical (H3.1)-like and 3 'replication-independent' (H3.3)-like forms, and encoding six distinct H3 isoforms. Transcripts for H3.3 genes including H3F3A show gradual decrease in abundance associated with developmental progression, whereas H3.1 transcripts including HIST1H3B tend to be strongly downregulated at an early prenatal stage and remain essentially silent thereafter. Twelve genes, including members of both H3.1 and H3.3 classes, contain a K27-AAG codon that is mutable to that for M (ATG), whereas the remaining contain the alternative, AAA codon for K at this position. H3F3A is the only H3.3-like gene containing the K27-AAG codon, whereas HIST1H3B is among ten H3.1-like genes containing this codon. This data indicates that, in the early developing human brain, HIST1H3B constitutes the largest proportion of H3.1 transcripts among H3.1 isoforms. We suggest that the apparent overrepresentation of K27M mutations in H3F3A relative to other H3 isoforms may result from its uniqueness among H3.3s for the K27-AAG codon and the functional relationship between H3.3 and PRC2, whereas overrepresentation of K27M mutations in HIST1H3B may be a product of strong relative expression of this gene in the early developing brain.

Expression of Placental Members of the Human Growth Hormone Gene Family Is Increased in Response to Sequential Inhibition of DNA Methylation and Histone Deacetylation.

The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V, are located at a single locus on chromosome 17. Efficient expression of these placental genes has been linked to local regulatory (5′ P and 3′ enhancer) sequences and a remote locus control region (LCR), in part, through gene transfer in placental and nonplacental tumor cells. However, low levels of endogenous hCS/GH-V transcripts are reported in the same cells compared with term placenta, suggesting that chromatin structure, or regulatory region accessibility, versus transcription factor availability contributes to the relatively low levels. To assess individual hCS-A, CS-B, and GH-V gene expression in placental and nonplacental tumor cells and the effect of increasing chromatin accessibility by inhibiting DNA methylation and histone deacetylation using 5-aza-2′-deoxycytidine (azadC) and trichostatin A (TSA). Low levels of hCS-A, CS-B, and GH-V were detected in placental and nonplacental tumor cells compared with term placenta. A significant >5-fold increase in activity was seen in placental, but not nonplacental, cells transfected with hybrid hCS promoter luciferase genes containing 3′ enhancer sequences. Pretreatment of placental JEG-3 cells with azadC resulted in a >10-fold increase in hCS-A, CS-B, and GH-V RNA levels with TSA treatment compared with TSA treatment alone. This effect was specific as reversing the treatment regimen did not have the same effect. An assessment of hyperacetylated H3/H4 in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure, including the hCS 3′ enhancer sequences and LCR. These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3′ enhancer sequences to available placental enhancer transcription factors.

Retraction Note to: Genetic variations in the KIR gene family may contribute to susceptibility to ankylosing spondylitis: a meta-analysis.

The present meta-analysis of relevant case–control studies was conducted to investigate the possible relationships between genetic variations in the killer cell immunoglobulin-like receptor (KIR) gene clusters of the human KIR gene family and susceptibility to ankylosing spondylitis (AS). The following electronic databases were searched for relevant articles without language restrictions: the Web of Science, the Cochrane Library Database, PubMed, EMBASE, CINAHL, the Chinese Biomedical Database (CBM) and Chinese National Knowledge Infrastructure (CNKI) databases, covering all papers published until 2013. STATA statistical software was adopted in this meta-analysis as well. We also calculated the crude odds ratios (OR) and its 95 % confidence intervals (95 % CI). Seven case–control studies with 1,004 patients diagnosed with AS and 2,138 healthy cases were implicated in our meta-analysis, and 15 genes in the KIR gene family were also evaluated. The results of our meta-analysis show statistical significance between the genetic variations in the KIR2DL1, KIR2DS4, KIR2DS5 and KIR3DS1 genes and an increased susceptibility to AS (KIR2DL1: OR 7.82, 95 % CI 3.87–15.81, P< 0.001; KIR2DS4: OR 1.91, 95 % CI 1.16–3.13, P = 0.010; KIR2DS5: OR1.51, 95 % CI 1.14–2.01, P = 0.004; KIR3DS1: OR 1.58, 95 % CI 1.34–1.86, P< 0.001; respectively). However, we failed to found positive correlations between other genes and susceptibility to AS (all P >0.05). The current meta-analysis provides reliable evidence that genetic variations in the KIR gene family may contribute to susceptibility to AS, especially for the KIR2DL1, KIR2DS4, KIR2DS5 and KIR3DS1 genes.

Abstract 2141: Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells

Abstract In this study we investigated cellular and molecular mechanisms for breast cancer signaling by EGFR and Ras proteins, independent of activating mutations. Epidermal Growth Factor (EGF) ligation to its receptor, EGFR, at the plasma membrane (PM) activates Ras small GTPases, propagating Ras mitogenic signaling. However, activating Ras mutations are not associated with breast cancers. EGFR and palmitoylated (Pa-) Ras localize to lipid rafts at the PM. Pa-Ras isotypes (H- and N-Ras, but not K-Ras) cycle through vesicle trafficking pathways between the Golgi and PM. We have found that anterograde H-Ras vesicles are sorted in the endocytic recycling compartment (ERC) into vesicles which contain the epithelial-specific transport mediator Rab25 (Rab11c), and Rab11/FIP1C (Rab-coupling protein, RCP). RCP couples Rab25 to vesicles to mediate vesicle anterograde transport. Rab25 and RCP expression are highly correlated with breast cancer progression, aggressiveness, and invasion and metastasis. Rab25, unique among the large family of human Rab genes, has been identified as a major marker and potential stimulator of breast cancers. RCP was recently shown to be a breast cancer driver oncogene, supporting the suggestion that RCP and Rab25 status may be significant clinical markers. Moreover, RCP stimulates H-Ras activation. Meta-analysis of human breast cancer cell line panels shows that while most do not harbor H- or N-Ras mutations, both RCP and Rab25 are highly over-expressed and levels correlate with resistance to EGF and Ras signaling inhibitors. We demonstrate that Rab25, RCP, H-Ras and EGFR co-localized in the ERC and in transport vesicles in MCF10A mammary epithelial cells, and that dominant negative (DN) Rab25 sequestered H-Ras and EGFR from the plasma membrane. DN Rab25 or Rab25 knockdown by shRNA inhibited EGF-induced EGFR phosphorylation, H-Ras activation, and ERK phosphorylation (ppERK), demonstrating a functional link between Rab25 activity and EGFR and H-Ras signaling. Both ER/PR-positive and -negative human breast invasive ductal carcinoma tissues showed elevated ppERK correlated with elevated Rab25 expression over normal breast tissue, whereas HER2+ tumor tissues had elevated ppERK even with low Rab25 expression. Thus, Rab25 and RCP regulate EGFR and Ras trafficking to the plasma membrane, required for mitogenic signaling and associated with tumorigenic effects, even in Ras (WT) cells. Citation Format: Allison B. Herman, Jeremy G.T. Wurtzel, Lawrence E. Goldfinger. Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2141. doi:10.1158/1538-7445.AM2015-2141

The Tumor Suppressor BCL7B Functions in the Wnt Signaling Pathway

Human BCL7 gene family consists of BCL7A, BCL7B, and BCL7C. A number of clinical studies have reported that BCL7 family is involved in cancer incidence, progression, and development. Among them, BCL7B, located on chromosome 7q11.23, is one of the deleted genes in patients with Williams-Beuren syndrome. Although several studies have suggested that malignant diseases occurring in patients with Williams-Beuren syndrome are associated with aberrations in BCL7B, little is known regarding the function of this gene at the cellular level. In this study, we focused on bcl-7, which is the only homolog of BCL7 gene family in Caenorhabditis elegans, and analyzed bcl-7 deletion mutants. As a result, we found that bcl-7 is required for the asymmetric differentiation of epithelial seam cells, which have self-renewal properties as stem cells and divide asymmetrically through the WNT pathway. Distal tip cell development, which is regulated by the WNT pathway in Caenorhabditis elegans, was also affected in bcl-7-knockout mutants. Interestingly, bcl-7 mutants exhibited nuclear enlargement, reminiscent of the anaplastic features of malignant cells. Furthermore, in KATOIII human gastric cancer cells, BCL7B knockdown induced nuclear enlargement, promoted the multinuclei phenotype and suppressed cell death. In addition, this study showed that BCL7B negatively regulates the Wnt-signaling pathway and positively regulates the apoptotic pathway. Taken together, our data indicate that BCL7B/BCL-7 has some roles in maintaining the structure of nuclei and is involved in the modulation of multiple pathways, including Wnt and apoptosis. This study may implicate a risk of malignancies with BCL7B-deficiency, such as Williams-Beuren syndrome.

Type 1 Diabetes High-Risk HLA Class II Determination by Polymerase Chain Reaction Sequence-Specific Primers.

The only strategy to select individuals at increased risk for type 1 diabetes for primary prevention trials is through genetic risk assessment. While genome-wide association studies have identified more than 40 loci associated with type 1 diabetes, the single most important genetic determinants lie within the human leucocyte antigen gene family on chromosome 6.In this chapter we describe a protocol for a straightforward, cheap strategy to determine HLA class II mediated risk of type 1 diabetes. This method has proved robust for genotyping whole-genome-amplified DNA as well as DNA extracted directly from human tissues.