Shelley Force Aldred

Polymorphisms within the C-reactive protein (CRP) promoter region are associated with plasma CRP levels.

Elevated plasma levels of C-reactive protein (CRP), an inflammation-sensitive marker, have emerged as an important predictor of future cardiovascular disease and metabolic abnormalities in apparently healthy men and women. Here, we performed a systematic survey of common nucleotide variation across the genomic region encompassing the CRP gene locus. Of the common single-nucleotide polymorphisms (SNPs) identified, several in the CRP promoter region are strongly associated with CRP levels in a large cohort study of cardiovascular risk in European American and African American young adults. We also demonstrate the functional importance of these SNPs in vitro.

Functional analysis of transcription factor binding sites in human promoters

BackgroundThe binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells. To characterize transcription factor binding site function on a large scale, we predicted and mutagenized 455 binding sites in human promoters. We carried out functional tests on these sites in four different immortalized human cell lines using transient transfections with a luciferase reporter assay, primarily for the transcription factors CTCF, GABP, GATA2, E2F, STAT, and YY1.ResultsIn each cell line, between 36% and 49% of binding sites made a functional contribution to the promoter activity; the overall rate for observing function in any of the cell lines was 70%. Transcription factor binding resulted in transcriptional repression in more than a third of functional sites. When compared with predicted binding sites whose function was not experimentally verified, the functional binding sites had higher conservation and were located closer to transcriptional start sites (TSSs). Among functional sites, repressive sites tended to be located further from TSSs than were activating sites. Our data provide significant insight into the functional characteristics of YY1 binding sites, most notably the detection of distinct activating and repressing classes of YY1 binding sites. Repressing sites were located closer to, and often overlapped with, translational start sites and presented a distinctive variation on the canonical YY1 binding motif.ConclusionsThe genomic properties that we found to associate with functional TF binding sites on promoters -- conservation, TSS proximity, motifs and their variations -- point the way to improved accuracy in future TFBS predictions.

Sequence features that drive human promoter function and tissue specificity

Promoters are important regulatory elements that contain the necessary sequence features for cells to initiate transcription. To functionally characterize a large set of human promoters, we measured the transcriptional activities of 4575 putative promoters across eight cell lines using transient transfection reporter assays. In parallel, we measured gene expression in the same cell lines and observed a significant correlation between promoter activity and endogenous gene expression (r = 0.43). As transient transfection assays directly measure the promoting effect of a defined fragment of DNA sequence, decoupled from epigenetic, chromatin, or long-range regulatory effects, we sought to predict whether a promoter was active using sequence features alone. CG dinucleotide content was highly predictive of ubiquitous promoter activity, necessitating the separation of promoters into two groups: high CG promoters, mostly ubiquitously active, and low CG promoters, mostly cell line-specific. Computational models trained on the binding potential of transcriptional factor (TF) binding motifs could predict promoter activities in both high and low CG groups: average area under the receiver operating characteristic curve (AUC) of the models was 91% and exceeded the AUC of CG content by an average of 23%. Known relationships, for example, between HNF4A and hepatocytes, were recapitulated in the corresponding cell lines, in this case the liver-derived cell line HepG2. Half of the associations between tissue-specific TFs and cell line-specific promoters were new. Our study underscores the importance of collecting functional information from complementary assays and conditions to understand biology in a systematic framework.

Two-tiered Approach Identifies a Network of Cancer and Liver Disease-related Genes Regulated by miR-122

MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.

A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays

BackgroundRegulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells.ResultsIn analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1.ConclusionThese data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.

Genome-Wide Discovery of Drug-Dependent Human Liver Regulatory Elements

Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

ABSTRACT Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

High-throughput microRNA target screening: miR-122 case study

Introduction MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes such as cellular signaling (1), cell differentiation, growth, development, and apoptosis (2). Mutations and improper regulation of miRNAs have been linked to a variety of physiological disorders such as cancer and heart disease (3,4). In animals, miRNAs are usually complementary to one or more a sites in the 3’UTRs of specific genes. Although current computational predictions of miRNA-UTR interactions provide important guidance for experimental analysis of miRNAs, little functional data exists on which to train prediction algorithms. Genome-wide transcript analysis can identify candidate target transcripts but cannot measure both the changes in a transcript’s stability or translational efficiency attributable to miRNAs. We have created a genome-wide library of human 3’UTR-luciferase reporter constructs to enable researchers to screen thousands of potential miRNA targets in a high throughput fashion. Using this strategy, we sought to identify new targets of miR-122, an important regulator of cholesterol and fatty-acid metabolism in liver that has been suggested as a therapeutic target for metabolic disease (5).

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Abstract LB-090: Sequence-based discovery of fully human anti-CD3 and anti-PDL1 single domain antibodies using novel transgenic rats

We have created a new platform and method for discovering fully human single domain antibodies (UniDabs) by combining novel transgenic rats (UniRats), antibody repertoire sequencing, and high-throughput gene assembly, expression and screening. UniRats express a comprehensive human VH gene repertoire in a heavy chain only antibody format, and they mount robust immune responses to a variety of antigens. UniRats do not produce endogenous Ig as their heavy, kappa and lambda loci have been silenced, so the only Ig expression comes from the human heavy chain transgenes. Human VHs identified using high-throughput functional screening of heavy chain antibodies may also be converted to a single domain UniDab format. UniDabs are powerful and flexible modular binding domains that may be combined to form multivalent structures, nanoparticle conjugates, or antibody-drug conjugates. In our preclinical CD3 and PDL1 programs, we have identified hundreds of antigen-specific UniDabs. This large collection of UniDabs covers a diverse set of epitopes, a wide range of affinities, and is a valuable resource for creating next-generation multi-specific cancer therapeutics. Citation Format: Nathan Trinklein, Shelley Force Aldred, Ute Schellenberger, Kat Harris, Starlynn Clarke, Kevin Dang, Duy Pham, Marianne Bruggemann, Wim vanSchooten. Sequence-based discovery of fully human anti-CD3 and anti-PDL1 single domain antibodies using novel transgenic rats. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-090.