Center for Genomics and Personalized Medicine

: Allele-specific Control Of Replication Timing And Genome Organization During Development; DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus x castaneus mouse crosses and exploited the high single-nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq), and chromatin accessibility (ATAC-seq). We also present HARP, a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/ 6,129 /sv, and CAST/ Ei), parental configurations, and gender revealed significant RT asynchrony between alleles across similar to 12% of the autosomal genome linked to subspecies genomes but not to parental origin, growth conditions, or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not as strongly with SNP density, gene expression, imprinting, or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types, including extraembryonic endoderm stem (XEN) cells, four male and female primary mouse embryonic fibroblasts (MEFs), and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs were largely lost in all differentiated cell types, accompanied by novel sites of allelic asynchrony at a considerably smaller proportion of the genome, suggesting that genome organization of homologs converges to similar folding patterns during cell fate commitment., Keywords: mouse, dna-replication, embryonic stem-cells, in-situ hybridization, hi-c, stable units, acute lymphoblastic-leukemia, asynchronous replication, es cells, x-chromosome inactivation, Publication Note: The publisher’s version of record is available at https://doi.org/10.1101/gr.232561.117

: Chromatin structure profile data from DNS-seq; Presented here are data from Next-Generation Sequencing of differential micrococcal nuclease digestions of formaldehyde-crosslinked chromatin in selected tissues of maize () inbred line B73. Supplemental materials include a wet-bench protocol for making DNS-seq libraries, the DNS-seq data processing pipeline for producing genome browser tracks. This report also includes the peak-calling pipeline using the iSeg algorithm to segment positive and negative peaks from the DNS-seq difference profiles. The data repository for the sequence data is the NCBI SRA, BioProject Accession 8., Grant Number: R01 GM126558, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117953.

: DNA replication timing alterations identify common markers between distinct progeroid diseases.; Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease. Here, we characterized DNA RT program alterations in Hutchinson-Gilford progeria syndrome (HGPS) and Rothmund-Thomson syndrome (RTS) patients compared with natural aging and cellular senescence. Our results identified a progeroid-specific RT signature that is common to cells from three HGPS and three RTS patients and distinguishes them from healthy individuals across a wide range of ages. Among the RT abnormalities, we identified the tumor protein p63 gene () as a gene marker for progeroid syndromes. By using the redifferentiation of four patient-derived induced pluripotent stem cells as a model for the onset of progeroid syndromes, we tracked the progression of RT abnormalities during development, revealing altered RT of the gene as an early event in disease progression of both HGPS and RTS. Moreover, the RT abnormalities in progeroid patients were associated with altered isoform expression of Our findings demonstrate the value of RT studies to identify biomarkers not detected by other methods, reveal abnormal RT as an early event in progeroid disease progression, and suggest gene regulation as a potential therapeutic target., Keywords: DNA replication timing, RT signatures, TP63, Progeroid diseases, Grant Number: P01 GM085354, R01 GM083337, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754778.

: Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish; The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljr(p23ahub)) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljr(p23ahub) as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljr(p23ahub). Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation across species., Keywords: cell fate specification, color-vision, cone-rod dystrophy, embryonic zebrafish, eye development, homeobox gene, nocturnal bottleneck, nuclear receptor, opsin genes, visual pigments, Publication Note: The publisher’s version of record is available at http://www.dx.doi.org/10.1371/journal.pgen.1005968

: Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish.; The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation across species., Grant Number: F31 EY020106, R01 EY017753, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825938.

: Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.; This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R., Grant Number: P01 GM085354, R01 GM083337, U54 DK107965, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044726.

: Hpsc-derived Tissue-resident Macrophage Model Reveals Differential Responses Of Macrophages To Zikv And Deny Infection; Zika virus (ZIKV) and dengue virus (DENV) are two closely related flaviviruses that lead to different clinical outcomes. The mechanism for the distinct pathogenesis of ZIKV and DENV is poorly understood. Here, we investigate ZIKV and DENV infection of macrophages using a human pluripotent stem cell (hPSC)-derived macrophage model and discover key virus-specific responses. ZIKV and DENV productively infect hPSC-derived macrophages. DENV, but not ZIKV, infection of macrophages strongly activates macrophage migration inhibitory factor (MIF) secretion and decreases macrophage migration. Neutralization of MIF leads to improved migratory ability of DENV-infected macrophages. In contrast, ZIKV-infected macrophages exhibit prolonged migration and express low levels of pro-inflammatory cytokines and chemokines. Mechanistically, ZIKV disrupts the nuclear factor kappa B (NF-kappa B)-MIF positive feedback loop by inhibiting the NF-kappa B signaling pathway. Our results demonstrate the utility of hPSC-derived macrophages in infectious disease modeling and suggest that the distinct impact of ZIKV and DENV on macrophage immune response may underlie different pathogenesis of Zika and dengue diseases., Keywords: activation, cells, disease, expression, mice, blood, nf-kappa-b, virus, dengue, migration inhibitory factor, Publication Note: The publisher’s version of record is available at https://doi.org/10.1016/j.stemcr.2018.06.006

: Iseg; Background: Identification of functional elements of a genome often requires dividing a sequence of measurements along a genome into segments where adjacent segments have different properties, such as different mean values. Despite dozens of algorithms developed to address this problem in genomics research, methods with improved accuracy and speed are still needed to effectively tackle both existing and emerging genomic and epigenomic segmentation problems. Results: We designed an efficient algorithm, called iSeg, for segmentation of genomic and epigenomic profiles. iSeg first utilizes dynamic programming to identify candidate segments and test for significance. It then uses a novel data structure based on two coupled balanced binary trees to detect overlapping significant segments and update them simultaneously during searching and refinement stages. Refinement and merging of significant segments are performed at the end to generate the final set of segments. By using an objective function based on the p-values of the segments, the algorithm can serve as a general computational framework to be combined with different assumptions on the distributions of the data. As a general segmentation method, it can segment different types of genomic and epigenomic data, such as DNA copy number variation, nucleosome occupancy, nuclease sensitivity, and differential nuclease sensitivity data. Using simple t-tests to compute p-values across multiple datasets of different types, we evaluate iSeg using both simulated and experimental datasets and show that it performs satisfactorily when compared with some other popular methods, which often employ more sophisticated statistical models. Implemented in C++, iSeg is also very computationally efficient, well suited for large numbers of input profiles and data with very long sequences. Conclusions: We have developed an efficient general-purpose segmentation tool and showed that it had comparable or more accurate results than many of the most popular segment-calling algorithms used in contemporary genomic data analysis. iSeg is capable of analyzing datasets that have both positive and negative values. Tunable parameters allow users to readily adjust the statistical stringency to best match the biological nature of individual datasets, including widely or sparsely mapped genomic datasets or those with non-normal distributions., Keywords: gene-expression, identification, design, high-resolution, array cgh data, chromatin reveals, circular binary segmentation, dna copy number, hidden markov model, maize genome, Publication Note: The publisher’s version of record is available at https://doi.org/10.1186/s12859-018-2140-3

: Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells.; The development of dengue antivirals and vaccine has been hampered by the incomplete understanding of molecular mechanisms of dengue virus (DENV) infection and pathology, partly due to the limited suitable cell culture or animal models that can capture the comprehensive cellular changes induced by DENV. In this study, we differentiated human pluripotent stem cells (hPSCs) into hepatocytes, one of the target cells of DENV, to investigate various aspects of DENV-hepatocyte interaction. hPSC-derived hepatocyte-like cells (HLCs) supported persistent and productive DENV infection. The activation of interferon pathways by DENV protected bystander cells from infection and protected the infected cells from massive apoptosis. Furthermore, DENV infection activated the NF-κB pathway, which led to production of proinflammatory cytokines and downregulated many liver-specific genes such as albumin and coagulation factor V. Our study demonstrates the utility of hPSC-derived hepatocytes as an in vitro model for DENV infection and reveals important aspects of DENV-host interactions., Grant Number: R21 AI119530, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031989.

: Open chromatin reveals the functional maize genome.; Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome., Keywords: Biased gene conversion, Chromatin, Maize, Recombination, Variance partitioning, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896728.

: Regulatory Landscape Of Early Maize Inflorescence Development; Background The functional genome of agronomically important plant species remains largely unexplored, yet presents a virtually untapped resource for targeted crop improvement. Functional elements of regulatory DNA revealed through profiles of chromatin accessibility can be harnessed for fine-tuning gene expression to optimal phenotypes in specific environments. Result Here, we investigate the non-coding regulatory space in the maize (Zea mays) genome during early reproductive development of pollen- and grain-bearing inflorescences. Using an assay for differential sensitivity of chromatin to micrococcal nuclease (MNase) digestion, we profile accessible chromatin and nucleosome occupancy in these largely undifferentiated tissues and classify at least 1.6% of the genome as accessible, with the majority of MNase hypersensitive sites marking proximal promoters, but also 3 ' ends of maize genes. This approach maps regulatory elements to footprint-level resolution. Integration of complementary transcriptome profiles and transcription factor occupancy data are used to annotate regulatory factors, such as combinatorial transcription factor binding motifs and long non-coding RNAs, that potentially contribute to organogenesis, including tissue-specific regulation between male and female inflorescence structures. Finally, genome-wide association studies for inflorescence architecture traits based solely on functional regions delineated by MNase hypersensitivity reveals new SNP-trait associations in known regulators of inflorescence development as well as new candidates. Conclusions These analyses provide a comprehensive look into thecis-regulatory landscape during inflorescence differentiation in a major cereal crop, which ultimately shapes architecture and influences yield potential., gene-expression, reveals, Accessible chromatin, class-i, dna elements, enhancer, Gene regulation, genome, lncRNAs, long noncoding rnas, Maize inflorescence, Meristem, open chromatin, tassel, transcription factor, The publisher's version of record is availible at https://doi.org/10.1186/s13059-020-02070-8

: Replicating Large Genomes; Complete duplication of large metazoan chromosomes requires thousands of potential initiation sites, only a small fraction of which are selected in each cell cycle. Assembly of the replication machinery is highly conserved and tightly regulated during the cell cycle, but the sites of initiation are highly flexible, and their temporal order of firing is regulated at the level of large-scale multi-replicon domains. Importantly, the number of replication forks must be quickly adjusted in response to replication stress to prevent genome instability. Here we argue that large genomes are divided into domains for exactly this reason. Once established, domain structure abrogates the need for precise initiation sites and creates a scaffold for the evolution of other chromosome functions., Grant Number: P01 GM085354, R01 GM083337, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893193.

: Stability of Wake-Sleep Cycles Requires Robust Degradation of the PERIOD Protein.; Robustness in biology is the stability of phenotype under diverse genetic and/or environmental perturbations. The circadian clock has remarkable stability of period and phase that-unlike other biological oscillators-is maintained over a wide range of conditions. Here, we show that the high fidelity of the circadian system stems from robust degradation of the clock protein PERIOD. We show that PERIOD degradation is regulated by a balance between ubiquitination and deubiquitination, and that disruption of this balance can destabilize the clock. In mice with a loss-of-function mutation of the E3 ligase gene β-Trcp2, the balance of PERIOD degradation is perturbed and the clock becomes dramatically unstable, presenting a unique behavioral phenotype unlike other circadian mutant animal models. We believe that our data provide a molecular explanation for how circadian phases, such as wake-sleep onset times, can become unstable in humans, and we present a unique mouse model to study human circadian disorders with unstable circadian rhythm phases., Keywords: USP14, Activity onset, Circadian, Deubiquitination, Period, Proteasomal degradation, Robustness, Sleep disorders, Ubiquitination, β-TRCP, Grant Number: R01 CA076584, R01 GM057587, R21 CA202200, R21 NS099813, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698108.

: Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia.; Genome-wide DNA replication timing (RT) profiles reflect the global three-dimensional chromosome architecture of cells. They also provide a comprehensive and unique megabase-scale picture of cellular epigenetic state. Thus, normal differentiation involves reproducible changes in RT, and transformation generally perturbs these, although the potential effects of altered RT on the properties of transformed cells remain largely unknown. A major challenge to interrogating these issues in human acute lymphoid leukemia (ALL) is the low proliferative activity of most of the cells, which may be further reduced in cryopreserved samples and difficult to overcome in vitro. In contrast, the ability of many human ALL cell populations to expand when transplanted into highly immunodeficient mice is well documented. To examine the stability of DNA RT profiles of serially passaged xenografts of primary human B- and T-ALL cells, we first devised a method that circumvents the need for bromodeoxyuridine incorporation to distinguish early versus late S-phase cells. Using this and more standard protocols, we found consistently strong retention in xenografts of the original patient-specific RT features. Moreover, in a case in which genomic analyses indicated changing subclonal dynamics in serial passages, the RT profiles tracked concordantly. These results indicate that DNA RT is a relatively stable feature of human ALLs propagated in immunodeficient mice. In addition, they suggest the power of this approach for future interrogation of the origin and consequences of altered DNA RT in ALL., Grant Number: P01 GM085354, R01 GM083337, R21 CA161666, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491210.

: hPSC-Derived Tissue-Resident Macrophage Model Reveals Differential Responses of Macrophages to ZIKV and DENV Infection.; Zika virus (ZIKV) and dengue virus (DENV) are two closely related flaviviruses that lead to different clinical outcomes. The mechanism for the distinct pathogenesis of ZIKV and DENV is poorly understood. Here, we investigate ZIKV and DENV infection of macrophages using a human pluripotent stem cell (hPSC)-derived macrophage model and discover key virus-specific responses. ZIKV and DENV productively infect hPSC-derived macrophages. DENV, but not ZIKV, infection of macrophages strongly activates macrophage migration inhibitory factor (MIF) secretion and decreases macrophage migration. Neutralization of MIF leads to improved migratory ability of DENV-infected macrophages. In contrast, ZIKV-infected macrophages exhibit prolonged migration and express low levels of pro-inflammatory cytokines and chemokines. Mechanistically, ZIKV disrupts the nuclear factor κB (NF-κB)-MIF positive feedback loop by inhibiting the NF-κB signaling pathway. Our results demonstrate the utility of hPSC-derived macrophages in infectious disease modeling and suggest that the distinct impact of ZIKV and DENV on macrophage immune response may underlie different pathogenesis of Zika and dengue diseases., Keywords: MIF, NF-κB signaling, Zika virus, Dengue virus, Disease modeling, Dissemination, Human pluripotent stem cells, Immune response, Macrophage differentiation, Macrophage migration, Grant Number: R21 AI119530, U19 AI131130, Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092684.

You are here

Center for Genomics and Personalized Medicine