A protocol for transposon insertion sequencing in Schizosaccharomyces pombe to identify factors that maintain heterochromatin.

Transposon insertion sequencing (TIS) is a highly effective method used with bacteria to identify genes important for growth in any condition of interest. Previously, we adapted this method to identify essential genes of the yeast Schizosaccharomyces pombe. Here, we describe modifications used to identify genes necessary for the formation of centromeric heterochromatin. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).

Non-canonical Targets of HIF1a Impair Oligodendrocyte Progenitor Cell Function.

Mammalian cells respond to insufficient oxygen through transcriptional regulators called hypoxia-inducible factors (HIFs). Although transiently protective, prolonged HIF activity drives distinct pathological responses in different tissues. Using a model of chronic HIF1a accumulation in pluripotent-stem-cell-derived oligodendrocyte progenitors (OPCs), we demonstrate that HIF1a activates non-canonical targets to impair generation of oligodendrocytes from OPCs. HIF1a activated a unique set of genes in OPCs through interaction with the OPC-specific transcription factor OLIG2. Non-canonical targets, including Ascl2 and Dlx3, were sufficient to block differentiation through suppression of the oligodendrocyte regulator Sox10. Chemical screening revealed that inhibition of MEK/ERK signaling overcame the HIF1a-mediated block in oligodendrocyte generation by restoring Sox10 expression without affecting canonical HIF1a activity. MEK/ERK inhibition also drove oligodendrocyte formation in hypoxic regions of human oligocortical spheroids. This work defines mechanisms by which HIF1a impairs oligodendrocyte formation and establishes that cell-type-specific HIF1a targets perturb cell function in response to low oxygen.

Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease.

Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD)1,2. Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD1,3-5. Here we show, using CRISPR-Cas9 to suppress Plp1 expression in the jimpy (Plp1jp) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders.

Dense Transposon Integration Reveals Essential Cleavage and Polyadenylation Factors Promote Heterochromatin Formation.

Heterochromatin functions as a scaffold for factors responsible for gene silencing and chromosome segregation. Heterochromatin can be assembled by multiple pathways, including RNAi and RNA surveillance. We identified factors that form heterochromatin using dense profiles of transposable element integration in Schizosaccharomyces pombe. The candidates include a large number of essential proteins such as four canonical mRNA cleavage and polyadenylation factors. We find that Iss1, a subunit of the poly(A) polymerase module, plays a role in forming heterochromatin in centromere repeats that is independent of RNAi. Genome-wide maps reveal that Iss1 accumulates at genes regulated by RNA surveillance. Iss1 interacts with RNA surveillance factors Mmi1 and Rrp6, and importantly, Iss1 contributes to RNA elimination that forms heterochromatin at meiosis genes. Our profile of transposable element integration supports the model that a network of mRNA cleavage and polyadenylation factors coordinates RNA surveillance, including the mechanism that forms heterochromatin at meiotic genes.

Mismatch repair-signature mutations activate gene enhancers across human colorectal cancer epigenomes.

Commonly-mutated genes have been found for many cancers, but less is known about mutations in cis-regulatory elements. We leverage gains in tumor-specific enhancer activity, coupled with allele-biased mutation detection from H3K27ac ChIP-seq data, to pinpoint potential enhancer-activating mutations in colorectal cancer (CRC). Analysis of a genetically-diverse cohort of CRC specimens revealed that microsatellite instable (MSI) samples have a high indel rate within active enhancers. Enhancers with indels show evidence of positive selection, increased target gene expression, and a subset is highly recurrent. The indels affect short homopolymer tracts of A/T and increase affinity for FOX transcription factors. We further demonstrate that signature mismatch-repair (MMR) mutations activate enhancers using a xenograft tumor metastasis model, where mutations are induced naturally via CRISPR/Cas9 inactivation of MLH1 prior to tumor cell injection. Our results suggest that MMR signature mutations activate enhancers in CRC tumor epigenomes to provide a selective advantage.

Corrigendum: Positively selected enhancer elements endow osteosarcoma cells with metastatic competence.

This corrects the article DOI: 10.1038/nm.4475.

Positively selected enhancer elements endow osteosarcoma cells with metastatic competence.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.

Serial number tagging reveals a prominent sequence preference of retrotransposon integration.

Transposable elements (TE) have both negative and positive impact on the biology of their host. As a result, a balance is struck between the host and the TE that relies on directing integration to specific genome territories. The extraordinary capacity of DNA sequencing can create ultra dense maps of integration that are being used to study the mechanisms that position integration. Unfortunately, the great increase in the numbers of insertion sites detected comes with the cost of not knowing which positions are rare targets and which sustain high numbers of insertions. To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions. We sequenced 1 million insertions of retrotransposon Tf1 in the genome of Schizosaccharomyces pombe and obtained the first profile of integration with frequencies for each individual position. Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration. The serial number system is a general method that can be applied to determine precise integration maps for retroviruses and gene therapy vectors.

Integration profiling of gene function with dense maps of transposon integration.

Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average, we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.

Time-Dependent Effects of Pre-Aging 3D Polymer Scaffolds in Cell Culture Medium on Cell Proliferation.

Protein adsorption is known to direct biological response to biomaterials and is important in determining cellular response in tissue scaffolds. In this study we investigated the effect of the duration of protein adsorption to 3D polymer scaffolds on cell attachment and proliferation. 3D macro-porous polymer scaffolds were pre-aged in serum-containing culture medium for 5 min, 1 d or 7 d prior to seeding osteoblasts. The total amount of protein adsorbed was found to increase with pre-ageing time. Cell attachment and proliferation were measured 1 d and 14 d, respectively, after cell seeding. Osteoblast proliferation, but not attachment, increased with scaffold pre-ageing time and amount of adsorbed serum protein. These results demonstrate that the amount of time that scaffolds are exposed to serum-containing medium can affect cell proliferation and suggest that these effects are mediated by differences in the amount of protein adsorption.

Stabilizing labile DNA-protein complexes in polyacrylamide gels.

The electrophoretic mobility-shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. EMSA, as standardly practiced today, works well for complexes with association binding constants K(a)>10(9) M(-1) under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.