Over-Expression of DSCAM and COL6A2 Cooperatively Generates Congenital Heart Defects

A significant current challenge in human genetics is the identification of interacting genetic loci mediating complex polygenic disorders. One of the best characterized polygenic diseases is Down syndrome (DS), which results from an extra copy of part or all of chromosome 21. A short interval near the distal tip of chromosome 21 contributes to congenital heart defects (CHD), and a variety of indirect genetic evidence suggests that multiple candidate genes in this region may contribute to this phenotype. We devised a tiered genetic approach to identify interacting CHD candidate genes. We first used the well vetted Drosophila heart as an assay to identify interacting CHD candidate genes by expressing them alone and in all possible pairwise combinations and testing for effects on rhythmicity or heart failure following stress. This comprehensive analysis identified DSCAM and COL6A2 as the most strongly interacting pair of genes. We then over-expressed these two genes alone or in combination in the mouse heart. While over-expression of either gene alone did not affect viability and had little or no effect on heart physiology or morphology, co-expression of the two genes resulted in ≈50% mortality and severe physiological and morphological defects, including atrial septal defects and cardiac hypertrophy. Cooperative interactions between DSCAM and COL6A2 were also observed in the H9C2 cardiac cell line and transcriptional analysis of this interaction points to genes involved in adhesion and cardiac hypertrophy. Our success in defining a cooperative interaction between DSCAM and COL6A2 suggests that the multi-tiered genetic approach we have taken involving human mapping data, comprehensive combinatorial screening in Drosophila, and validation in vivo in mice and in mammalian cells lines should be applicable to identifying specific loci mediating a broad variety of other polygenic disorders

Simulation of immiscible multiphase flow in porous media using a moving finite element algorithm.

A moving grid finite element method is developed to solve the nonlinear coupled set of partial differential equations (PDEs) governing immiscible multiphase flow in porous media in one dimension. This is one of the first research works to develop an adaptive grid algorithm to solve a coupled set of nonlinear PDEs. The model's algorithm is based on a time implicit st and ard Galerkin finite element method, with linear basis functions and an iterative Picard procedure. Grid adaptation may be accomplished after each iteration, every time step or every few time steps. The adaptation method is based upon a single equidistribution equation involving gradient and curvature criteria. This single equidistribution equation is employed in place of the more complex alternative which involves solving a multiobjective distribution problem, applying distribution criteria to all the PDEs variables. The simplification is achieved by assigning the largest gradient at each node as a representative gradient. Use of a single reduced equidistribution equation facilitates the incorporation of the adaptive grid algorithm in the numerical solver for a coupled set of nonlinear PDEs. This work includes sensitivity analyses for the parameters which are incorporated in the grid adaptation method, including curvature weighting parameters, artificial viscosity and artificial repulsive force coefficients. Consideration is also given to methods for preserving phase mass in the grid adaptation process. The FEM used herein incorporates a FEM chord slope analog for the saturation derivatives with respect to the capillary pressures to avoid oscillations in the solution of the capillary pressures and phase volume balance. This work includes a discussion of the implementation of a fixed organic chemical pressure boundary condition. The discussion shows that division of the boundary pressure into two parts, one which is applied to the boundary capillary pressure, and one which is applied to the nonorganic fluid boundary pressure to force this phase to flow away from the boundary, generates results similar to physical observations. The model is verified against the finite difference model of Abriola (1984). Its applicability and versatility are demonstrated through solution of complex two- and three-phase flow problems involving single and double fronts and a sink/source term. The model is shown to achieve significant savings in computation time and memory allocation, when compared with fixed grid solutions of equivalent accuracy.Ph.D.Civil engineeringPetroleum engineeringMathematicsUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/162307/1/9001630.pd

Long-distance association of topological boundaries through nuclear condensates

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates

Signal-induced enhancer activation requires Ku70 to read topoisomerase1–DNA covalent complexes

Enhancer activation serves as the main mechanism regulating signal-dependent transcriptional programs, ensuring cellular plasticity, yet central questions persist regarding their mechanism of activation. Here, by successfully mapping topoisomerase I-DNA covalent complexes genome-wide, we find that most, if not all, acutely activated enhancers, including those induced by 17β-estradiol, dihydrotestosterone, tumor necrosis factor alpha and neuronal depolarization, are hotspots for topoisomerase I-DNA covalent complexes, functioning as epigenomic signatures read by the classic DNA damage sensor protein, Ku70. Ku70 in turn nucleates a heterochromatin protein 1 gamma (HP1γ)-mediator subunit Med26 complex to facilitate acute, but not chronic, transcriptional activation programs. Together, our data uncover a broad, unappreciated transcriptional code, required for most, if not all, acute signal-dependent enhancer activation events in both mitotic and postmitotic cells

Dismissal of RNA Polymerase II Underlies a Large Ligand-Induced Enhancer Decommissioning Program

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers

Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17β-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation