Computational Inference of Genome-Wide Protein-DNA Interactions Using High-Throughput Genomic Data

Transcriptional regulation has been studied intensively in recent decades. One important aspect of this regulation is the interaction between regulatory proteins, such as transcription factors (TF) and nucleosomes, and the genome. Different high-throughput techniques have been invented to map these interactions genome-wide, including ChIP-based methods (ChIP-chip, ChIP-seq, etc.), nuclease digestion methods (DNase-seq, MNase-seq, etc.), and others. However, a single experimental technique often only provides partial and noisy information about the whole picture of protein-DNA interactions. Therefore, the overarching goal of this dissertation is to provide computational developments for jointly modeling different experimental datasets to achieve a holistic inference on the protein-DNA interaction landscape.

We first present a computational framework that can incorporate the protein binding information in MNase-seq data into a thermodynamic model of protein-DNA interaction. We use a correlation-based objective function to model the MNase-seq data and a Markov chain Monte Carlo method to maximize the function. Our results show that the inferred protein-DNA interaction landscape is concordant with the MNase-seq data and provides a mechanistic explanation for the experimentally collected MNase-seq fragments. Our framework is flexible and can easily incorporate other data sources. To demonstrate this flexibility, we use prior distributions to integrate experimentally measured protein concentrations.

We also study the ability of DNase-seq data to position nucleosomes. Traditionally, DNase-seq has only been widely used to identify DNase hypersensitive sites, which tend to be open chromatin regulatory regions devoid of nucleosomes. We reveal for the first time that DNase-seq datasets also contain substantial information about nucleosome translational positioning, and that existing DNase-seq data can be used to infer nucleosome positions with high accuracy. We develop a Bayes-factor-based nucleosome scoring method to position nucleosomes using DNase-seq data. Our approach utilizes several effective strategies to extract nucleosome positioning signals from the noisy DNase-seq data, including jointly modeling data points across the nucleosome body and explicitly modeling the quadratic and oscillatory DNase I digestion pattern on nucleosomes. We show that our DNase-seq-based nucleosome map is highly consistent with previous high-resolution maps. We also show that the oscillatory DNase I digestion pattern is useful in revealing the nucleosome rotational context around TF binding sites.

Finally, we present a state-space model (SSM) for jointly modeling different kinds of genomic data to provide an accurate view of the protein-DNA interaction landscape. We also provide an efficient expectation-maximization algorithm to learn model parameters from data. We first show in simulation studies that the SSM can effectively recover underlying true protein binding configurations. We then apply the SSM to model real genomic data (both DNase-seq and MNase-seq data). Through incrementally increasing the types of genomic data in the SSM, we show that different data types can contribute complementary information for the inference of protein binding landscape and that the most accurate inference comes from modeling all available datasets.

This dissertation provides a foundation for future research by taking a step toward the genome-wide inference of protein-DNA interaction landscape through data integration.

HDAC inhibitors cause site-specific chromatin remodeling at PU.1-bound enhancers in K562 cells

BACKGROUND: Small molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies. However, clinical use is often confounded by toxicity, perhaps due to indiscriminate hyperacetylation of cellular proteins. Therefore, elucidating the mechanisms by which HDACi trigger differentiation, cell cycle arrest, or apoptosis of cancer cells could inform development of more targeted therapies. We used the myelogenous leukemia line K562 as a model of HDACi-induced differentiation to investigate chromatin accessibility (DNase-seq) and expression (RNA-seq) changes associated with this process. RESULTS: We identified several thousand specific regulatory elements [~10 % of total DNase I-hypersensitive (DHS) sites] that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid treatment. Most of the differential DHS sites display hallmarks of enhancers, including being enriched for non-promoter regions, associating with nearby gene expression changes, and increasing luciferase reporter expression in K562 cells. Differential DHS sites were enriched for key hematopoietic lineage transcription factor motifs, including SPI1 (PU.1), a known pioneer factor. We found PU.1 increases binding at opened DHS sites with HDACi treatment by ChIP-seq, but PU.1 knockdown by shRNA fails to block the chromatin accessibility and expression changes. A machine-learning approach indicates H3K27me3 initially marks PU.1-bound sites that open with HDACi treatment, suggesting these sites are epigenetically poised. CONCLUSIONS: We find HDACi treatment of K562 cells results in site-specific chromatin remodeling at epigenetically poised regulatory elements. PU.1 shows evidence of a pioneer role in this process by marking poised enhancers but is not required for transcriptional activation.

The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoretic-mobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.

Numerical modelling of solder joint formation

Solder materials are used to provide a connection between electronic components and printed circuit boards (PCBs) using either the reflow or wave soldering process. As a board assembly passes through a reflow furnace the solder (initially in the form of solder paste) melts, reflows, then solidifies, and finally deforms between the chip and board. A number of defects may occur during this process such as flux entrapment, void formation, and cracking of the joint, chip or board. These defects are a serious concern to industry, especially with trends towards increasing component miniaturisation and smaller pitch sizes. This paper presents a modelling methodology for predicting solder joint shape, solidification, and deformation (stress) during the assembly process.

Exploiting virtual prototyping for reliability assessment

Products manufactured by the electronics sector are having a major impact in telecommunications, transportation space applications, biomedical applications, consumer products, intelligent hand held devices, and of course,the computer. Demands from end-users in terms of greater product functionality, adoption of environmentally friendly materials, and further miniaturization continually pose several challenges to electronics companies. In the context of electronic product design and manufacture, virtual prototying software tools are allowing companies to dramatically reduce the number of phsysical prototypes and design iterations required in product development and hence reduce costs and time to market. This paper details of the trends in these technolgies and provides an example of their use for flip-chip assembly technology.

Optimization and finite element analysis for reliable electronic packaging

This paper demonstrates a modeling and design approach that couples computational mechanics techniques with numerical optimisation and statistical models for virtual prototyping and testing in different application areas concerning reliability of eletronic packages. The integrated software modules provide a design engineer in the electronic manufacturing sector with fast design and process solutions by optimizing key parameters and taking into account complexity of certain operational conditions. The integrated modeling framework is obtained by coupling the multi-phsyics finite element framework - PHYSICA - with the numerical optimisation tool - VisualDOC into a fully automated design tool for solutions of electronic packaging problems. Response Surface Modeling Methodolgy and Design of Experiments statistical tools plus numerical optimisaiton techniques are demonstrated as a part of the modeling framework. Two different problems are discussed and solved using the integrated numerical FEM-Optimisation tool. First, an example of thermal management of an electronic package on a board is illustrated. Location of the device is optimized to ensure reduced junction temperature and stress in the die subject to certain cooling air profile and other heat dissipating active components. In the second example thermo-mechanical simulations of solder creep deformations are presented to predict flip-chip reliability and subsequently used to optimise the life-time of solder interconnects under thermal cycling.

Modelling the effect of temperature on product reliability

Reliability of electronic parts is a major concern for many manufacturers, since early failures in the field can cost an enormous amount to repair - in many cases far more than the original cost of the product. A great deal of effort is expended by manufacturers to determine the failure rates for a process or the fraction of parts that will fail in a period of time. It is widely recognized that the traditional approach to reliability predictions for electronic systems are not suitable for today's products. This approach, based on statistical methods only, does not address the physics governing the failure mechanisms in electronic systems. This paper discusses virtual prototyping technologies which can predict the physics taking place and relate this to appropriate failure mechanisms. Simulation results illustrate the effect of temperature on the assembly process of an electronic package and the lifetime of a flip-chip package.

Modelling the metal arc weldpool

Traditionally, before flip chips can be assembled the dies have to be attached with solder bumps. This process involves the deposition of metal layers on the Al pads on the dies and this is called the under bump metallurgy (UBM). In an alternative process, however, Copper (Cu) columns can be used to replace solder bumps and the UBM process may be omitted altogether. After the bumping process, the bumped dies can be assembled on to the printed circuit board (PCB) by using either solder or conductive adhesives. In this work, the reliability issues of flip chips with Cu column bumped dies have been studied. The flip chip lifetime associated with the solder fatigue failure has been modeled for a range of geometric parameters. The relative importance of these parameters is given and solder volume has been identified as the most important design parameter for long-term reliability. Another important problem that has been studied in this work is the dissolution of protection metals on the pad and Cu column in the reflow process. For small solder joints the amount of Cu which dissolves into the molten solder after the protection layers have worn out may significantly affect solder joint properties.

Computational modeling techniques for reliability of electronic components on printed circuit boards

This paper describes modeling technology and its use in providing data governing the assembly and subsequent reliability of electronic chip components on printed circuit boards (PCBs). Products, such as mobile phones, camcorders, intelligent displays, etc., are changing at a tremendous rate where newer technologies are being applied to satisfy the demands for smaller products with increased functionality. At ever decreasing dimensions, and increasing number of input/output connections, the design of these components, in terms of dimensions and materials used, is playing a key role in determining the reliability of the final assembly. Multiphysics modeling techniques are being adopted to predict a range of interacting physics-based phenomena associated with the manufacturing process. For example, heat transfer, solidification, marangoni fluid flow, void movement, and thermal-stress. The modeling techniques used are based on finite volume methods that are conservative and take advantage of being able to represent the physical domain using an unstructured mesh. These techniques are also used to provide data on thermal induced fatigue which is then mapped into product lifetime predictions.

Correlation of solder paste rheology with computational simulations of the stencil printing process

Soldering technologies continue to evolve to meet the demands of the continuous miniaturisation of electronic products, particularly in the area of solder paste formulations used in the reflow soldering of surface mount devices. Stencil printing continues to be a leading process used for the deposition of solder paste onto printed circuit boards (PCBs) in the volume production of electronic assemblies, despite problems in achieving a consistent print quality at an ultra-fine pitch. In order to eliminate these defects a good understanding of the processes involved in printing is important. Computational simulations may complement experimental print trials and paste characterisation studies, and provide an extra dimension to the understanding of the process. The characteristics and flow properties of solder pastes depend primarily on their chemical and physical composition and good material property data is essential for meaningful results to be obtained by computational simulation.This paper describes paste characterisation and computational simulation studies that have been undertaken through the collaboration of the School of Aeronautical, Mechanical and Manufacturing Engineering at Salford University and the Centre for Numerical Modelling and Process Analysis at the University of Greenwich. The rheological profile of two different paste formulations (lead and lead-free) for sub 100 micron flip-chip devices are tested and applied to computational simulations of their flow behaviour during the printing process.

Reliability predictions for high density packaging

Predicting the reliability of newly designed products, before manufacture, is obviously highly desirable for many organisations. Understanding the impact of various design variables on reliability allows companies to optimise expenditure and release a package in minimum time. Reliability predictions originated in the early years of the electronics industry. These predictions were based on historical field data which has evolved into industrial databases and specifications such as the famous MIL-HDBK-217 standard, plus numerous others. Unfortunately the accuracy of such techniques is highly questionable especially for newly designed packages. This paper discusses the use of modelling to predict the reliability of high density flip-chip and BGA components. A number of design parameters are investigated at the assembly stage, during testing, and in-service.

Thermal, mechanical and optical modelling of VCSEL packaging

Hybrid OECB (Opto-Electrical Circuit Boards) are expected to make a significant impact in the telecomm switches arena within the next five years, creating optical backplanes with high speed point-to-point optical interconnects. OECB's incorporate short range optical interconnects, and are based on VCSEL (Vertical Cavity Surface Emitting Diode) and PD (Photo Diode) pairs, connected to each other via embedded waveguides in the OECB. The VCSEL device is flip-chip assembled onto an organic substrate with embedded optical waveguides. The performance of the VCSEL device is governed by the thermal, mechanical and optical characteristics of this assembly. During operation, the VCSEL device will heat up and the thermal change together with the CTE mismatch in the materials, will result in potential misalignment between the VCSEL apertures and the waveguide openings in the substrate. Any degree of misalignment will affect the optical performance of the package. This paper will present results from a highly coupled modelling analysis involving thermal, mechanical and optical models. The paper will also present results from an optimisation analysis based on Design of Experiments (DOE).

Mathematical modelling: a laser soldering process for an optoelectronics butterfly package

For sensitive optoelectronic components, traditional soldering techniques cannot be used because of their inherent sensitivity to thermal stresses. One such component is the Optoelectronic Butterfly Package which houses a laser diode chip aligned to a fibre-optic cable. Even sub-micron misalignment of the fibre optic and laser diode chip can significantly reduce the performance of the device. The high cost of each unit requires that the number of damaged components, via the laser soldering process, are kept to a minimum. Mathematical modelling is undertaken to better understand the laser soldering process and to optimize operational parameters such as solder paste volume, copper pad dimensions, laser solder times for each joint, laser intensity and absorption coefficient. Validation of the model against experimental data will be completed, and will lead to an optimization of the assembly process, through an iterative modelling cycle. This will ultimately reduce costs, improve the process development time and increase consistency in the laser soldering process.

Computer modelling of the reliability of flip chips with metal column bumping

Recently, research has been carried out to test a novel bumping method which omits the under bump metallurgy forming process by bonding copper columns directly onto the Al pads of the silicon dies. This bumping method could be adopted to simplify the flip chip manufacturing process, increase the productivity and achieve a higher I/O count. This paper describes an investigation of the solder joint reliability of flip-chips based on this new bumping process. Computer modelling methods are used to predict the shape of solder joints and response of flip chips to thermal cyclic loading. The accumulated plastic strain energy at the comer solder joints is used as the damage indicator. Models with a range of design parameters have been compared for their reliability. The parameters that have been investigated are the copper column height, radius and solder volume. The ranking of the relative importance of these parameters is given. For most of the results presented in the paper, the solder material has been assumed to be the lead-free 96.5Sn3.5Ag alloy but some results for 60Sn40Pb solder joints have also been presented.

Response Surface Modeling and Optimisation for Reliable Electronic Products

The aim of integrating computational mechanics (FEA and CFD) and optimization tools is to speed up dramatically the design process in different application areas concerning reliability in electronic packaging. Design engineers in the electronics manufacturing sector may use these tools to predict key design parameters and configurations (i.e. material properties, product dimensions, design at PCB level. etc) that will guarantee the required product performance. In this paper a modeling strategy coupling computational mechanics techniques with numerical optimization is presented and demonstrated with two problems. The integrated modeling framework is obtained by coupling the multi-physics analysis tool PHYSICA - with the numerical optimization package - Visua/DOC into a fuJly automated design tool for applications in electronic packaging. Thermo-mechanical simulations of solder creep deformations are presented to predict flip-chip reliability and life-time under thermal cycling. Also a thermal management design based on multi-physics analysis with coupled thermal-flow-stress modeling is discussed. The Response Surface Modeling Approach in conjunction with Design of Experiments statistical tools is demonstrated and used subsequently by the numerical optimization techniques as a part of this modeling framework. Predictions for reliable electronic assemblies are achieved in an efficient and systematic manner.