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Genome-wide association studies have identified FGFR2 as a breast cancer (BC) susceptibility gene in populations of European and Asian descent, but a causative variant has not yet been conclusively identified. We hypothesized that the weaker linkage disequilibrium across this associated region in populations of African ancestry might help refine the set of candidate-causal single nucleotide polymorphisms (SNPs) previously identified by our group. Eight candidate-causal SNPs were evaluated in 1253 African American invasive BC cases and 1245 controls. A significant association with BC risk was found with SNP rs2981578 (unadjusted per-allele odds ratio = 1.20, 95% confidence interval 1.03-1.41, P(trend) = 0.02), with the odds ratio estimate similar to that reported in European and Asian subjects. To extend the fine-mapping, genotype data from the African American studies were analyzed jointly with data from European (n = 7196 cases, 7275 controls) and Asian (n = 3901 cases, 3205 controls) studies. In the combined analysis, SNP rs2981578 was the most strongly associated. Five other SNPs were too strongly correlated to be excluded at a likelihood ratio of < 1/100 relative to rs2981578. Analysis of DNase I hypersensitive sites indicated that only two of these map to highly accessible chromatin, one of which, SNP rs2981578, has previously been implicated in up-regulating FGFR2 expression. Our results demonstrate that the association of SNPs in FGFR2 with BC risk extends to women of African American ethnicity, and illustrate the utility of combining association analysis in datasets of diverse ethnic groups with functional experiments to identify disease susceptibility variants.

The recent whole-genome scan for breast cancer has revealed the FGFR2 (fibroblast growth factor receptor 2) gene as a locus associated with a small, but highly significant, increase in the risk of developing breast cancer. Using fine-scale genetic mapping of the region, it has been possible to narrow the causative locus to a haplotype of eight strongly linked single nucleotide polymorphisms (SNPs) spanning a region of 7.5 kilobases (kb) in the second intron of the FGFR2 gene. Here we describe a functional analysis to define the causative SNP, and we propose a model for a disease mechanism. Using gene expression microarray data, we observed a trend of increased FGFR2 expression in the rare homozygotes. This trend was confirmed using real-time (RT) PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028. To elucidate which SNPs might be responsible for this difference, we examined protein–DNA interactions for the eight most strongly disease-associated SNPs in different breast cell lines. We identify two cis-regulatory SNPs that alter binding affinity for transcription factors Oct-1/Runx2 and C/EBPβ, and we demonstrate that both sites are occupied in vivo. In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression. We propose a model in which the Oct-1/Runx2 and C/EBPβ binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation., Author Summary Recently, a number of whole-genome association studies have identified genes that predispose individuals to common diseases such as cancer. The challenge now is to understand how the identified risk loci contribute to disease, since the majority of these loci are located within introns (which are discarded after transcription) and intergenic regions, and therefore do not change the coding region of nearby genes. This manuscript describes how two single–base pair changes in intron 2 of the FGFR2 (fibroblast growth factor receptor 2) gene, “the top hit” of the breast cancer susceptibility study, exert their function. We find that the changes alter the binding of two transcription factors and cause an increase in FGFR2 gene expression, thus providing a molecular explanation for the risk phenotype. This is the first functional study, to our knowledge, of the risk loci identified for breast cancer in a whole-genome scan and demonstrates that these studies can be used as valid starting points for studying the underlying biology of cancer., Recent whole-genome scans have identified novel risk genes for many common diseases, challenging researchers to determine how these genes contribute to disease. A new study provides molecular insights into a breast cancer risk factor.

The first enhancers to be identified in the immunoglobulin gene loci are located in the J-C intron. However, deletion of the immunoglobulin kappa intron-enhancer has little effect on the transcription of kappa transgenes. Here we ask whether the second kappa enhancer which we recently identified at the 3'-end of the locus plays a role in kappa gene expression. We show that its omission leads to 20-40 fold lower expression of kappa transgenes and to poor allelic exclusion. Transfection experiments show that activity of the 3'-enhancer, like that of the kappa-intron enhancer, can be induced in a pre-B cell line by incubation with bacterial lipopolysaccharide. Whereas induction of the kappa-intron enhancer is due to induction of NF-kappa B activity, deletion mapping of the 3'-enhancer localises its activity to a 50 nucleotide region that lacks an NF-kappa B site; indeed the 3'-enhancer allows kappa expression in a cell line which lacks NF-kappa B. Thus, both the 3'- and intron-enhancers can be induced at the same stage of differentiation but by distinct pathways. Furthermore, unlike the intron-enhancer, the 3'-enhancer plays a critical role in the transcription of rearranged immunoglobulin kappa genes.

Expression of the rearranged chicken immunoglobulin light chain (IgL) gene is regulated by a V gene promoter, a matrix attachment region (MAR) in the J-C intron and an enhancer downstream of the Ig constant region. Using knockout analysis, we demonstrate that the 3′-enhancer is not only required for gene activation but is also essential for the maintenance of gene expression. Deletion of the MAR on the other hand increases IgL transcription, indicating that the MAR acts as negative regulator. We demonstrate that Id1 and Id3, dominant-negative regulators of basic-region helix-loop-helix (bHLH) transcription factors, are able to reduce chicken IgL 3′-enhancer activity in transient assays and strongly reduce the rate of gene conversion (GC) in DT40 clone 18 cells. Conversely, overexpression of avian E47, a bHLH transcription factor, leads to a dramatic increase in GC rates independent of IgL or activation-induced cytidine deaminase RNA levels. Thus, E47 is the first transcription factor to activate GC without an apparent increase in transcription.

Balancing cell death and proliferation plays a key role in the maintenance of homeostasis and the prevention of cancer in all multicellullar organisms. The Bcl-2 family of proteins regulates programmed cell death and plays an important role in the selection of lymphocytes (reviewed in1). It comprises pro-apoptotic (Bcl-2 and Bcl-x) and antiapoptotic members (Bak, Bax and Bim), whose coordinate activities regulate opposing pathways of proliferation and cell death. The bcl-2 gene has long been associated with tumorigenesis in B lineage cells. Indeed it derives its name from its location at the interchromosomal breakpoint t14;18 which is the molecular hallmark of follicular B cell lymphoma. The translocated bcl-2 gene is overexpressed causing an increase in cell survival, thereby promoting the development of follicular lymphoma. Similarly, overexpression of bcl-2 in transgenic mice leads to a polyclonal expansion of B cells, again mediated by increased survival but not increased proliferation. Upon accumulation of secondary mutations these mice develop malignant lymphoma, demonstrating the contribution of Bcl-2 to tumorigenesis.2 Overexpression of Bcl-2 due to gene amplification has also been implicated in the aetiology of B-cell lymphoma.3 It is thus not surprising that the regulation of bcl-2 gene expression has been studied in some detail.

Genome-wide association studies (GWASs) have identified thousands of single nucleotide polymorphisms (SNPs) associated with human traits and diseases. But because the vast majority of these SNPs are located in the noncoding regions of the genome their risk promoting mechanisms are elusive. Employing a new methodology combining cistromics, epigenomics and genotype imputation we annotate the noncoding regions of the genome in breast cancer cells and systematically identify the functional nature of SNPs associated with breast cancer risk. Our results demonstrate that breast cancer risk-associated SNPs are enriched in the cistromes of FOXA1 and ESR1 and the epigenome of H3K4me1 in a cancer and cell-type-specific manner. Furthermore, the majority of these risk-associated SNPs modulate the affinity of chromatin for FOXA1 at distal regulatory elements, which results in allele-specific gene expression, exemplified by the effect of the rs4784227 SNP on the TOX3 gene found within the 16q12.1 risk locus.

Six years after the publication of the first breast cancer GWAS, skepticism is expressed about the usefulness of the results. The GWAS loci identified to date explain only a minority of the estimated genetic variance, and the risks conferred by even the strongest individual variants are small. Classical biochemical pathway approaches to elucidating mechanism have had modest success. I will argue that an attempt to examine the combined effects by common and rare variants and their interactions through analysis based on gene regulatory networks may yield new and useful information. Although still speculative, I will present initial data that provide support for this approach. Citation Format: Bruce A. J. Ponder, Kerstin Meyer, Michael Fletcher, Florian Markowetz, Ines de Santiago, Mauro Castro. Gene regulatory network approaches to interpreting breast cancer GWAS results. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA01. doi:10.1158/1538-7445.CANSUSC14-IA01

Ig kappa gene expression is controlled by two enhancers, one located within the major intron (Ei) and the other located downstream of C kappa (E3'). Whereas loss of E3' has previously been shown to diminish kappa expression, we show here that a rearranged kappa transgene lacking Ei is well expressed, even at the pre-B cell stage. This suggests that E3' alone might be sufficient to give properly regulated transcription throughout B cell development. Indeed, we show that a transgene composed of a beta-globin reporter linked to E3' is expressed in a B cell-specific manner, becoming activated at the late pro-B to pre-B cell stage but with dramatically enhanced activity on B cell activation. Thus, E3' becomes active as a transcription enhancer at the stage when V kappa-J kappa rearrangement is being initiated and is sufficient to yield an expression pattern in a linked reporter gene similar to that of fully rearranged kappa genes.

Both the kappa intron and the kappa 3' enhancer are required for high levels of immunoglobulin kappa gene expression. The activity of both enhancer elements can be induced by LPS in pre-B cells. While the LPS induction of the kappa intron enhancer is mediated by NF-kappa B, this factor is not responsible for activation of the 3' enhancer. Dissection of the 3' enhancer has shown that in pre-B cells the activity of the kappa 3' enhancer is repressed by a region flanking an active core element. We have now scanned this flanking region for nuclear factor binding sites and have identified sites for B-cell specific E47/E12-like proteins and two ubiquitous nuclear proteins. Furthermore, we have identified a nuclear factor in pre-B cells whose binding activity is suppressed in response to LPS. In its tissue-distribution and binding specificity this factor appears to be identical to the lymphoid specific protein LEF-1. The position of the LEF-1 binding site within the 3' enhancer and its response to LPS raise the possibility that LEF-1 may be the target for a second pathway able to mediate LPS induction of immunoglobulin kappa gene transcription.

During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of E2A. Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47. The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion.

The description of cell lines capable of transcribing immunoglobulin heavy or light chain genes in the apparent absence of an active enhancer has led us to look for novel enhancers in the immunoglobulin gene loci. Here we show that there is a second B-cell-specific enhancer in the mouse kappa locus and that this is located 9 kb downstream of C kappa. This enhancer is some 7-fold stronger than the kappa-intron enhancer and shows striking sequence homologies to the lymphotropic papovavirus, IgH and kappa-intron enhancers. The location of the kappa 3' enhancer between C kappa and the RS element means that it is deleted in some B cells that express lambda light chains.

SummaryHuman lungs enable efficient gas exchange, and form an interface with the environment which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in health. We report location-dependent airway epithelial cell states, and a novel subset of tissue-resident memory T cells. In lower airways of asthma patients, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report presence of pathogenic effector Th2 cells in asthma, and find evidence for type-2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identify a shift from airway structural cell communication in health to a Th2-dominated interactome in asthma.

Visualization and analysis of molecular networks are both central to systems biology. However, there still exists a large technological gap between them, especially when assessing multiple network levels or hierarchies. Here we present RedeR, an R/Bioconductor package combined with a Java core engine for representing modular networks. The functionality of RedeR is demonstrated in two different scenarios: hierarchical and modular organization in gene co-expression networks and nested structures in time-course gene expression subnetworks. Our results demonstrate RedeR as a new framework to deal with the multiple network levels that are inherent to complex biological systems. RedeR is available from http://bioconductor.org/packages/release/bioc/html/RedeR.html.