Daniel L. Vera

Topologically associating domains are stable units of replication-timing regulation

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400–800 kilobases (‘replication domains’), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program

Mammalian genomes are partitioned into domains that replicate in a defined temporal order. These domains can replicate at similar times in all cell types (constitutive) or at cell type-specific times (developmental). Genome-wide chromatin conformation capture (Hi-C) has revealed sub-megabase topologically associating domains (TADs), which are the structural counterparts of replication domains. Hi-C also segregates inter-TAD contacts into defined 3D spatial compartments that align precisely to genome-wide replication timing profiles. Determinants of the replication-timing program are re-established during early G1 phase of each cell cycle and lost in G2 phase, but it is not known when TAD structure and inter-TAD contacts are re-established after their elimination during mitosis. Here, we use multiplexed 4C-seq to study dynamic changes in chromatin organization during early G1. We find that both establishment of TADs and their compartmentalization occur during early G1, within the same time frame as establishment of the replication-timing program. Once established, this 3D organization is preserved either after withdrawal into quiescence or for the remainder of interphase including G2 phase, implying 3D structure is not sufficient to maintain replication timing. Finally, we find that developmental domains are less well compartmentalized than constitutive domains and display chromatin properties that distinguish them from early and late constitutive domains. Overall, this study uncovers a strong connection between chromatin re-organization during G1, establishment of replication timing, and its developmental control.

Open chromatin reveals the functional maize genome

Significance The maize genome, similar to those of most plant genomes, is 98% noncoding. Much of the remainder is a vast desert of repeats that remain repressed throughout the cell cycle. The plant cell orchestrates its complex activities by restricting access to functional regions with an open chromatin configuration. Here, we identify the small portion (<1%) of the maize genome residing in open chromatin. We demonstrate that open chromatin predicts molecular phenotypes such as gene expression and recombination. Furthermore, we show that genetic variation within open chromatin regions accounts for ∼40% of phenotypic variation in agronomic traits. By greatly narrowing the scope of the functional maize genome, this study can help to accelerate the pace of crop improvement through highly focused genomic selection and genome editing. 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.

Differential nuclease sensitivity profiling of chromatin reveals biochemical footprints coupled to gene expression and functional DNA elements in maize.

This work presents a technique to identify specific regions of maize chromatin that are hypersensitive to digestion by micrococcal nuclease, which preferentially cleaves DNA not bound into nucleosomes. This chromatin profiling technique helps elucidate how global changes in gene expression relate to changes in nucleosome position and should be broadly applicable to other species. The eukaryotic genome is organized into nucleosomes, the fundamental units of chromatin. The positions of nucleosomes on DNA regulate protein-DNA interactions and in turn influence DNA-templated events. Despite the increasing number of genome-wide maps of nucleosome position, how global changes in gene expression relate to changes in nucleosome position is poorly understood. We show that in nucleosome occupancy mapping experiments in maize (Zea mays), particular genomic regions are highly susceptible to variation introduced by differences in the extent to which chromatin is digested with micrococcal nuclease (MNase). We exploited this digestion-linked variation to identify protein footprints that are hypersensitive to MNase digestion, an approach we term differential nuclease sensitivity profiling (DNS-chip). Hypersensitive footprints were enriched at the 5′ and 3′ ends of genes, associated with gene expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the transcription factor KNOTTED1. We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints. These results reveal biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements. This approach to chromatin profiling should be broadly applicable to other species and should shed light on the relationships among chromatin organization, protein-DNA interactions, and genome regulation.

Genome-Wide Prediction of Nucleosome Occupancy in Maize Reveals Plant Chromatin Structural Features at Genes and Other Elements at Multiple Scales

Genome-wide prediction of nucleosome occupancy in maize was performed using a computational approach, and the predictions were validated using biochemical data and reveal novel features of maize promoters. The nucleosome is a fundamental structural and functional chromatin unit that affects nearly all DNA-templated events in eukaryotic genomes. It is also a biochemical substrate for higher order, cis-acting gene expression codes and the monomeric structural unit for chromatin packaging at multiple scales. To predict the nucleosome landscape of a model plant genome, we used a support vector machine computational algorithm trained on human chromatin to predict the nucleosome occupancy likelihood (NOL) across the maize (Zea mays) genome. Experimentally validated NOL plots provide a novel genomic annotation that highlights gene structures, repetitive elements, and chromosome-scale domains likely to reflect regional gene density. We established a new genome browser (http://www.genomaize.org) for viewing support vector machine-based NOL scores. This annotation provides sequence-based comprehensive coverage across the entire genome, including repetitive genomic regions typically excluded from experimental genomics data. We find that transposable elements often displayed family-specific NOL profiles that included distinct regions, especially near their termini, predicted to have strong affinities for nucleosomes. We examined transcription start site consensus NOL plots for maize gene sets and discovered that most maize genes display a typical +1 nucleosome positioning signal just downstream of the start site but not upstream. This overall lack of a –1 nucleosome positioning signal was also predicted by our method for Arabidopsis (Arabidopsis thaliana) genes and verified by additional analysis of previously published Arabidopsis MNase-Seq data, revealing a general feature of plant promoters. Our study advances plant chromatin research by defining the potential contribution of the DNA sequence to observed nucleosome positioning and provides an invariant baseline annotation against which other genomic data can be compared.

Chromatin patterns associated with lung adenocarcinoma progression

The development and progression of lung adenocarcinoma, one of the most common cancers, is driven by the interplay of genetic and epigenetic changes and the role of chromatin structure in malignant transformation remains poorly understood. We used systematic nucleosome distribution and chromatin accessibility microarray mapping platforms to analyze the genome-wide chromatin structure from normal tissues and from primary lung adenocarcinoma of different grades and stages. We identified chromatin-based patterns across different patients with lung adenocarcinoma of different cancer grade and stage. Low-grade cancers had nucleosome distributions very different compared with the corresponding normal tissue but had nearly identical chromatin accessibility. Conversely, nucleosome distributions of high-grade cancers showed few differences. Substantial disruptions in chromosomal accessibility were seen in a patient with a high-grade and high-stage tumor. These data imply that chromatin structure changes during the progression of lung adenocarcinoma. We have therefore developed a model in which low-grade lung adenocarcinomas are linked to changes in nucleosome distributions, whereas higher-grade tumors are linked to large-scale chromosomal changes. These results provide a foundation for the development of a comprehensive framework linking the general and locus-specific roles of chromatin structure to lung cancer progression. We propose that this strategy has the potential to identify a new class of chromatin-based diagnostic, prognostic and therapeutic markers in cancer progression.

Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells

Summary 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.

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.

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.

QTL Mapping and Candidate Gene Analysis of Telomere Length Control Factors in Maize (Zea mays L.).

Telomere length is a quantitative trait important for many cellular functions. Failure to regulate telomere length contributes to genomic instability, cellular senescence, cancer, and apoptosis in humans, but the functional significance of telomere regulation in plants is much less well understood. To gain a better understanding of telomere biology in plants, we used quantitative trait locus (QTL) mapping to identify genetic elements that control telomere length variation in maize (Zea mays L.). For this purpose, we measured the median and mean telomere lengths from 178 recombinant inbred lines of the IBM mapping population and found multiple regions that collectively accounted for 33–38% of the variation in telomere length. Two-way analysis of variance revealed interaction between the quantitative trait loci at genetic bin positions 2.09 and 5.04. Candidate genes within these and other significant QTL intervals, along with select genes known a priori to regulate telomere length, were tested for correlations between expression levels and telomere length in the IBM population and diverse inbred lines by quantitative real-time PCR. A slight but significant positive correlation between expression levels and telomere length was observed for many of the candidate genes, but Ibp2 was a notable exception, showing instead a negative correlation. A rad51-like protein (TEL-MD_5.04) was strongly supported as a candidate gene by several lines of evidence. Our results highlight the value of QTL mapping plus candidate gene expression analysis in a genetically diverse model system for telomere research.