Profiling Chromatin Landscape at High Resolution and Throughput with 2C-ChIP.

Chromatin immunoprecipitation (ChIP) is used to probe the presence of proteins and/or their posttranslational modifications on genomic DNA. This method is often used alongside chromosome conformation capture approaches to obtain a better-rounded view of the functional relationship between chromatin architecture and its landscape. Since the inception of ChIP, its protocol has been modified to improve speed, sensitivity, and specificity. Combining ChIP with deep sequencing has recently improved its throughput and made genome-wide profiling possible. However, genome-wide analysis is not always the best option, particularly when many samples are required to study a given genomic region or when quantitative data is desired. We recently developed carbon copy-ChIP (2C-ChIP), a new form of the high-throughput ChIP analysis method ideally suited for these types of studies. 2C-ChIP applies ligation-mediated amplification (LMA) followed by deep sequencing to quantitatively detect specified genomic regions in ChIP samples. Here, we describe the generation of 2C-ChIP libraries and computational processing of the resulting sequencing data.

PRC2-Associated Chromatin Contacts in the Developing Limb Reveal a Possible Mechanism for the Atypical Role of PRC2 in HoxA Gene Expression.

Preventing inappropriate gene expression in time and space is as fundamental as triggering the activation of tissue- or cell-type-specific factors at the correct developmental stage and in the correct cells. Here, we study the impact of Polycomb repressive complex 2 (PRC2) function on HoxA gene regulation. We analyze chromatin conformation of the HoxA cluster and its regulatory regions and show that in addition to the well-known role of PRC2 in silencing Hox genes via direct binding, its function is required for the changes in HoxA long-range interactions distinguishing proximal limbs from distal limbs. This effect stems from the differential PRC2 occupancy over the HoxA cluster and, at least in part, from the ability of PRC2-bound loci to engage in long-range contacts. Unexpectedly, PRC2 also impacts chromatin conformation in a way that promotes enhancer-promoter contacts required for proper HoxA expression, pointing to a dual role of PRC2 in gene regulation.

2C-ChIP: measuring chromatin immunoprecipitation signal from defined genomic regions with deep sequencing.

BACKGROUND: Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking. RESULTS: Here, we present carbon copy-ChIP (2C-ChIP), a versatile, inexpensive, and high-throughput technique to quantitatively measure the abundance of DNA sequences in ChIP samples. This method combines ChIP with ligation-mediated amplification (LMA) and deep sequencing to probe large genomic regions of interest. 2C-ChIP recapitulates results from benchmark ChIP approaches. We applied 2C-ChIP to the HOXA cluster to find that a region where H3K27me3 and SUZ12 linger encodes HOXA-AS2, a long non-coding RNA that enhances gene expression during cellular differentiation. CONCLUSIONS: 2C-ChIP fills the need for a robust molecular biology tool designed to probe dedicated genomic regions in a high-throughput setting. The flexible nature of the 2C-ChIP approach allows rapid changes in experimental design at relatively low cost, making it a highly efficient method for chromatin analysis.

Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells.

Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9-donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9-donor conjugation in addition to delivery influence HDR efficiency.

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture.

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.

Clustering of tissue-specific sub-TADs accompanies the regulation of HoxA genes in developing limbs.

HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet "DNA loops". Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.

The small molecule GMX1778 is a potent inhibitor of NAD+ biosynthesis: strategy for enhanced therapy in nicotinic acid phosphoribosyltransferase 1-deficient tumors.

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD(+) biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD(+) turnover, which makes NAD(+) modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD(+) and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD(+) repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.