Discovery of cell-type specific regulatory elements in the human genome using differential chromatin modification analysis

Chromatin modifications have been comprehensively illustrated to play important roles in gene regulation and cell diversity in recent years. Given the rapid accumulation of genome-wide chromatin modification maps across multiple cell types, there is an urgent need for computational methods to analyze multiple maps to reveal combinatorial modification patterns and define functional DNA elements, especially those are specific to cell types or tissues. In this current study, we developed a computational method using differential chromatin modification analysis (dCMA) to identify cell-type-specific genomic regions with distinctive chromatin modifications. We then apply this method to a public data set with modification profiles of nine marks for nine cell types to evaluate its effectiveness. We found cell-type-specific elements unique to each cell type investigated. These unique features show significant cell-type-specific biological relevance and tend to be located within functional regulatory elements. These results demonstrate the power of a differential comparative epigenomic strategy in deciphering the human genome and characterizing cell specificity.     

Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells

Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated.     

TRAP-rc,Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.     

THUNDER: A reference-free deconvolution method to infer cell type proportions from bulk Hi-C data

Hi-C data provide population averaged estimates of three-dimensional chromatin contacts across cell types and states in bulk samples. Effective analysis of Hi-C data entails controlling for the potential confounding factor of differential cell type proportions across heterogeneous bulk samples. We propose a novel unsupervised deconvolution method for inferring cell type composition from bulk Hi-C data, the Two-step Hi-c UNsupervised DEconvolution appRoach (THUNDER). We conducted extensive simulations to test THUNDER based on combining two published single-cell Hi-C (scHi-C) datasets. THUNDER more accurately estimates the underlying cell type proportions compared to reference-free methods (e.g., TOAST, and NMF) and is more robust than reference-dependent methods (e.g. MuSiC). We further demonstrate the practical utility of THUNDER to estimate cell type proportions and identify cell-type-specific interactions in Hi-C data from adult human cortex tissue samples. THUNDER will be a useful tool in adjusting for varying cell type composition in population samples, facilitating valid and more powerful downstream analysis such as differential chromatin organization studies. Additionally, THUNDER estimated contact profiles provide a useful exploratory framework to investigate cell-type-specificity of the chromatin interactome while experimental data is still rare.     

Preferential Lentiviral Targeting of Astrocytes in the Central Nervous System

The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment.     

Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior

Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examining Drosophila melanogaster fru P1 neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. Additionally, we find chromatin modifications that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination in fru P1 neurons. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP). We identify actively translated genes in fru P1 neurons, revealing novel stage- and sex-differences in gene expression. We also find chromatin modification enrichment patterns that are associated with gene expression. Next, we use the chromatin modification data to identify cell-type-specific super-enhancer-containing genes. We show that genes with super-enhancers in fru P1 neurons differ across development and between the sexes. We validated that a set of genes are expressed in fru P1 neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression in fru P1 neurons.