GRaNIE and GRaNPA: inference and evaluation of enhancer-mediated gene regulatory networks.

Enhancers play a vital role in gene regulation and are critical in mediating the impact of noncoding genetic variants associated with complex traits. Enhancer activity is a cell-type-specific process regulated by transcription factors (TFs), epigenetic mechanisms and genetic variants. Despite the strong mechanistic link between TFs and enhancers, we currently lack a framework for jointly analysing them in cell-type-specific gene regulatory networks (GRN). Equally important, we lack an unbiased way of assessing the biological significance of inferred GRNs since no complete ground truth exists. To address these gaps, we present GRaNIE (Gene Regulatory Network Inference including Enhancers) and GRaNPA (Gene Regulatory Network Performance Analysis). GRaNIE (https://git.embl.de/grp-zaugg/GRaNIE) builds enhancer-mediated GRNs based on covariation of chromatin accessibility and RNA-seq across samples (e.g. individuals), while GRaNPA (https://git.embl.de/grp-zaugg/GRaNPA) assesses the performance of GRNs for predicting cell-type-specific differential expression. We demonstrate their power by investigating gene regulatory mechanisms underlying the response of macrophages to infection, cancer and common genetic traits including autoimmune diseases. Finally, our methods identify the TF PURA as a putative regulator of pro-inflammatory macrophage polarisation.

Upregulation of SPS100 gene expression by an antisense RNA via a switch of mRNA isoforms with different stabilities.

Pervasive transcription of genomes generates multiple classes of non-coding RNAs. One of these classes are stable long non-coding RNAs which overlap coding genes in antisense direction (asRNAs). The function of such asRNAs is not fully understood but several cases of antisense-dependent gene expression regulation affecting the overlapping genes have been demonstrated. Using high-throughput yeast genetics and a limited set of four growth conditions we previously reported a regulatory function for ∼25% of asRNAs, most of which repress the expression of the sense gene. To further explore the roles of asRNAs we tested more conditions and identified 15 conditionally antisense-regulated genes, 6 of which exhibited antisense-dependent enhancement of gene expression. We focused on the sporulation-specific gene SPS100, which becomes upregulated upon entry into starvation or sporulation as a function of the antisense transcript SUT169. We demonstrate that the antisense effect is mediated by its 3' intergenic region (3'-IGR) and that this regulation can be transferred to other genes. Genetic analysis revealed that SUT169 functions by changing the relative expression of SPS100 mRNA isoforms from a short and unstable transcript to a long and stable species. These results suggest a novel mechanism of antisense-dependent gene regulation via mRNA isoform switching.

Donor cell memory confers a metastable state of directly converted cells.

Generation of induced oligodendrocyte progenitor cells (iOPCs) from somatic fibroblasts is a strategy for cell-based therapy of myelin diseases. However, iOPC generation is inefficient, and the resulting iOPCs exhibit limited expansion and differentiation competence. Here we overcome these limitations by transducing an optimized transcription factor combination into a permissive donor phenotype, the pericyte. Pericyte-derived iOPCs (PC-iOPCs) are stably expandable and functionally myelinogenic with high differentiation competence. Unexpectedly, however, we found that PC-iOPCs are metastable so that they can produce myelination-competent oligodendrocytes or revert to their original identity in a context-dependent fashion. Phenotypic reversion of PC-iOPCs is tightly linked to memory of their original transcriptome and epigenome. Phenotypic reversion can be disconnected from this donor cell memory effect, and in vivo myelination can eventually be achieved by transplantation of O4+ pre-oligodendrocytes. Our data show that donor cell source and memory can contribute to the fate and stability of directly converted cells.

Genomic Rewiring of SOX2 Chromatin Interaction Network during Differentiation of ESCs to Postmitotic Neurons.

Cellular differentiation requires dramatic changes in chromatin organization, transcriptional regulation, and protein production. To understand the regulatory connections between these processes, we generated proteomic, transcriptomic, and chromatin accessibility data during differentiation of mouse embryonic stem cells (ESCs) into postmitotic neurons and found extensive associations between different molecular layers within and across differentiation time points. We observed that SOX2, as a regulator of pluripotency and neuronal genes, redistributes from pluripotency enhancers to neuronal promoters during differentiation, likely driven by changes in its protein interaction network. We identified ATRX as a major SOX2 partner in neurons, whose co-localization correlated with an increase in active enhancer marks and increased expression of nearby genes, which we experimentally confirmed for three loci. Collectively, our data provide key insights into the regulatory transformation of SOX2 during neuronal differentiation, and we highlight the significance of multi-omic approaches in understanding gene regulation in complex systems.

Lysine 4 of histone H3.3 is required for embryonic stem cell differentiation, histone enrichment at regulatory regions and transcription accuracy.

Mutations in enzymes that modify histone H3 at lysine 4 (H3K4) or lysine 36 (H3K36) have been linked to human disease, yet the role of these residues in mammals is unclear. We mutated K4 or K36 to alanine in the histone variant H3.3 and showed that the K4A mutation in mouse embryonic stem cells (ESCs) impaired differentiation and induced widespread gene expression changes. K4A resulted in substantial H3.3 depletion, especially at ESC promoters; it was accompanied by reduced remodeler binding and increased RNA polymerase II (Pol II) activity. Regulatory regions depleted of H3.3K4A showed histone modification alterations and changes in enhancer activity that correlated with gene expression. In contrast, the K36A mutation did not alter H3.3 deposition and affected gene expression at the later stages of differentiation. Thus, H3K4 is required for nucleosome deposition, histone turnover and chromatin remodeler binding at regulatory regions, where tight regulation of Pol II activity is necessary for proper ESC differentiation.

Protein Abundance Control by Non-coding Antisense Transcription.

Stable unannotated transcripts (SUTs), some of which overlap protein-coding genes in antisense direction, are a class of non-coding RNAs. While case studies have reported important regulatory roles for several of such RNAs, their general impact on protein abundance regulation of the overlapping gene is not known. To test this, we employed seamless gene manipulation to repress antisense SUTs of 162 yeast genes by using a unidirectional transcriptional terminator and a GFP tag. We found that the mere presence of antisense SUTs was not sufficient to influence protein abundance, that observed effects of antisense SUTs correlated with sense transcript start site overlap, and that the effects were generally weak and led to reduced protein levels. Antisense regulated genes showed increased H3K4 di- and trimethylation and had slightly lower than expected noise levels. Our results suggest that the functionality of antisense RNAs has gene and condition-specific components.

PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast.

Here, we report on a novel PCR targeting-based strategy called 'PCR duplication' that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure.

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of ß-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.