DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape

Introduction: Transposable element (TE) derived sequences comprise half of our genome and DNA methylome, and are presumed densely methylated and inactive. Examination of the genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues revealed unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the tissue type and their expression correlated strongly with hypomethylation of the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks including H3K4me1 and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and exhibited evidence for evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type-specific regulatory networks, and have acquired tissue-specific epigenetic regulation

Imaging Unique DNA Sequences in Individual Cells Using a CRISPR-Cas9-Based, Split Luciferase Biosensor

An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both in vitro and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such “always-on” probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this “turn-on” probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during ex vivo editing procedures and should be a useful platform for many other live cell DNA biosensing applications

Identification of an OCT4 and SRY regulatory module using integrated computational and experimental genomics approaches

ChIP-chip studies have revealed that many in vivo binding sites have a weak match to the consensus sequence for the transcription factor being analyzed. Possible explanations for these observations include (1) the in vitro-derived consensus site does not represent the in vivo binding site and/or (2) the factor is recruited to a weak binding site via interaction with another protein. To address these possibilities, we developed an approach (ChIPMotifs) that incorporates a bootstrap resampling method to statistically infer the optimal cutoff threshold for a position weight matrix (PWM) of a motif identified from ChIP-chip data by ab initio motif discovery programs. Using OCT4 ChIP-chip data and the ChIPMotifs approach, we first developed a refined OCT4 PWM. We then used the refined PWM and a ChIPModules approach to identify transcription factors colocalizing with OCT4 in Ntera2 testicular embryonal carcinoma cells. We found that the consensus binding site for SRY, a transcription factor critical for testis development, colocalizes with the OCT4 PWM. To further characterize the relationship between OCT4 and SRY, we performed ChIP-chip experiments with human promoter microarrays, and found that 49% of the top ∼1000 OCT4 target promoters were also bound by SRY. This analysis represents the first identification of SRY target promoters. Interestingly, we determined that promoters bound by OCT4 and SRY, but not those bound by SRY alone, were also bound by the transcriptional repressor KAP1. Our studies not only validate the ChIPMotifs and ChIPModules combinatorial approach but also identify a possible new regulatory partner of OCT4

Unexpected binding behaviors of bacterial Argonautes in human cells cast doubts on their use as targetable gene regulators

<div><p>Prokaryotic Argonaute proteins (pAgos) have been proposed as an alternative to the CRISPR/Cas9 platform for gene editing. Although Argonaute from <i>Natronobacterium gregoryi</i> (<i>Ng</i>Ago) was recently shown unable to cleave genomic DNA in mammalian cells, the utility of <i>Ng</i>Ago or other pAgos as a targetable DNA-binding platform for epigenetic editing has not been explored. In this report, we evaluated the utility of two prokaryotic Argonautes (<i>Ng</i>Ago and <i>Tt</i>Ago) as DNA-guided DNA-binding proteins. <i>Ng</i>Ago showed no meaningful binding to chromosomal targets, while <i>Tt</i>Ago displayed seemingly non-specific binding to chromosomal DNA even in the absence of guide DNA. The observed lack of DNA-guided targeting and unexpected guide-independent genome sampling under the conditions in this study provide evidence that these pAgos might be suitable for neither gene nor epigenome editing in mammalian cells.</p></div

The potential for high-precision targeting by pAgos.

<p>A. Targeting of CRISPR/Cas9 is limited by the requirement of a PAM site (green boxes). B. <i>Ng</i>Ago or <i>Tt</i>Ago might allow the precise targeting of features, such as transcription factor binding sites (black boxes). gRNA/gDNA, red lines.</p

h<i>Tt</i>Ago and h<i>Ng</i>Ago do not cleave genomic target sites.

<p>A. Schematic of human codon-optimized <i>Tt</i>Ago and <i>Ng</i>Ago proteins containing two nuclear localization signals (NLSs) and a 3xFlag epitope tag. Western blot analysis of h<i>Tt</i>Ago and h<i>Ng</i>Ago proteins in HEK293 cells using an antibody against the 3xFlag tag. Untransfected cells serve as a negative control (-). Ponceau staining was used as a loading control. B. Diagram illustrating <i>RPL13A</i> and <i>HER2</i> guide DNAs and genomic target site. Genomic target sites are indicated in blue and complementary ssDNA guides are indicated in red. C. Amplicon sequencing was carried out on HEK293 cells co-transfected with h<i>Tt</i>Ago or h<i>Ng</i>Ago expression plasmids with (+) or without (-) gDNAs to <i>RPL13A</i> and <i>HER2</i>. To increase the amount of gDNAs, cells were re-transfected with gDNAs 24 hours after the initial transfection (++). CRISPRESSO analysis confirms that h<i>Tt</i>Ago and h<i>Ng</i>Ago did not cause insertions or deletions (indels) under any of these conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193818#pone.0193818.s008" target="_blank">S5 Table</a>). As a control, HEK293 cells were co-transfected with Cas9 nuclease and gRNA expression plasmids targeting <i>RPL13A</i> and <i>HER2</i>. RNA-guided Cas9 displayed target site cleavage at the genomic <i>RPL13A</i> and <i>HER2</i> target sites. The percentage of sequence reads containing indels relative to the total number of sequence reads is plotted on the y-axis.</p

Endogenous mammalian histone H3.3 exhibits chromatin-related functions during development

Abstract Background The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. Results H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile. Conclusions Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development

Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells

We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A. We found that (1) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; (2) a gene is generally repressed by only one mechanism; and (3) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation