Cohesin is required for long-range enhancer action

The mammalian genome is organised into topologically associating domains (TADs) that are formed through the process of cohesin-driven loop extrusion and whose extent is constrained at TAD boundaries by orientation-dependent CTCF binding. The large regulatory landscapes of developmental genes frequently correspond to TADs, leading to the hypothesis that TADs and/or loop extrusion are important for enhancers to act on their cognate gene. However, it has proven hard to interpret the consequences of experimental disruption of TADs or loop-extrusion on gene regulation, in part because of the difficulty in distinguishing direct from indirect effects on enhancer-driven gene expression. By coupling acute protein degradation with synthetic activation by targeted transcription factor recruitment in mouse embryonic stem cells, here we show that cohesin, but not CTCF, is required for activation of a target gene by distant distal regulatory elements. Cohesin is not required for activation directly at the promoter or activation from an enhancer located closer to the gene. Our findings support the hypothesis that chromatin compaction mediated by cohesin-mediated loop extrusion allows for genes to be activated by regulatory elements that are located many hundreds of kilobases away in the linear genome but suggests that cohesin is dispensable for more genomically close enhancers.

Whole gene analysis of a genotype G29P[6] human rotavirus strain identified in Central African Republic

Objective Rotavirus A (RVA) remains the main causative agent of gastroenteritis in young children and the young of many mammalian and avian species. In this study we describe a RVA strain detected from a 6-month-old child from Central African Republic (CAR). Results We report the 11 open reading frame sequences of a G29-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 rotavirus strain, RVA/Human-wt/CAR/CAR91/2014/G29P[6]. Nine genes (VP1–VP3, VP6, NSP1–NSP5) shared 90–100% sequence similarities with genogroup 2 rotaviruses. Phylogenetically, backbone genes, except for VP3 and NSP4 genes, were linked with cognate gene sequences of human DS-1-like genogroup 2, hence their genetic origin. The VP3 and NSP4 genes, clustered genetically with both human and animal strains, an indication genetic reassortment human and animal RVA strains has taken place. The VP7 gene shared nucleotide (93–94%) and amino acid (95.5–96.7%) identities with Kenyan and Belgian human G29 strains, as well as to buffalo G29 strain from South Africa, while the VP4 gene most closely resembled P[6]-lineage I strains from Africa and Bangladesh (97%).

The Role of Nuclear Insulin and IGF1 Receptors in Metabolism and Cancer

Insulin (InsR) and insulin-like growth factor-1 (IGF1R) receptors mediate the metabolic and growth-promoting actions of insulin and IGF1/IGF2, respectively. Evidence accumulated in recent years indicates that, in addition to their typical cell-surface localization pattern and ligand-activated mechanism of action, InsR and IGF1R are present in the cell nucleus of both normal and transformed cells. Nuclear translocation seems to involve interaction with a small, ubiquitin-like modifier protein (SUMO-1), although this modification is not always a prerequisite. Nuclear InsR and IGF1R exhibit a number of biological activities that classically fit within the definition of transcription factors. These nuclear activities include, among others, sequence-specific DNA binding and transcriptional control. Of particular interest, nuclear IGF1R was capable of binding and stimulating its cognate gene promoter. The physiological relevance of this autoregulatory mechanism needs to be further investigated. In addition to its nuclear localization, studies have identified IGF1R in the Golgi apparatus, and this particular distribution correlated with a migratory phenotype. In summary, the newly described roles of InsR and IGF1R as gene regulators, in concert with their atypical pattern of subcellular distribution, add a further layer of complexity to traditional models of cell signaling. Furthermore, and in view of the emerging role of IGF1R as a potential therapeutic target, a better understanding of the mechanisms responsible for nuclear IGF1R transport and identification of IGF1R interactors might help optimize target directed therapies in oncology.

Long non-coding RNA IGF2-AS represses breast cancer tumorigenesis by epigenetically regulating IGF2

Long non-coding RNAs are a kind of endogenous ncRNAs with a length of more than 200 bp. Accumulating evidence suggests that long non-coding RNAs function as pivotal regulators in tumorigenesis and progression. However, their biological roles in breast cancer remain largely unknown. Here, we found that IGF2 antisense RNA (IGF2-AS) was significantly decreased in breast cancer tissues, cell lines, and plasma. Patients with low IGF2-AS were more likely to develop larger tumor size and later clinical stage. Overexpression of IGF2-AS evidently inhibited the proliferation and induced apoptosis of MCF-7 and T47D cells in vitro, as well as retarded tumor growth in vivo. Further investigation revealed that IGF2-AS inhibited the expression of its sense-cognate gene IGF2 in an epigenetic DNMT1-dependent manner, resulting in the inactivation of downstream oncogenic PI3K/AKT/mTOR signaling pathway. Enforced expression of IGF2 could significantly block the tumor inhibitory effect of IGF2-AS. Importantly, we found that IGF2-AS could be used as an effective biomarker for breast cancer diagnosis and prognosis. Taken together, our study indicates that IGF2-AS is a tumor suppressor in breast cancer, restoration of IGF2-AS may be a promising treatment for this fatal disease.

Circulating testican-2 is a podocyte-derived marker of kidney health

In addition to their fundamental role in clearance, the kidneys release select molecules into the circulation, but whether any of these anabolic functions provides insight on kidney health is unknown. Using aptamer-based proteomics, we characterized arterial (A)-to-renal venous (V) gradients for >1,300 proteins in 22 individuals who underwent invasive sampling. Although most of the proteins that changed significantly decreased from A to V, consistent with renal clearance, several were found to increase, the most significant of which was testican-2. To assess the clinical implications of these physiologic findings, we examined proteomic data in the Jackson Heart Study (JHS), an African-American cohort (n = 1,928), with replication in the Framingham Heart Study (FHS), a White cohort (n = 1,621). In both populations, testican-2 had a strong, positive correlation with estimated glomerular filtration rate (eGFR). In addition, higher baseline testican-2 levels were associated with a lower rate of eGFR decline in models adjusted for age, gender, hypertension, type 2 diabetes, body mass index, baseline eGFR, and albuminuria. Glomerular expression of testican-2 in human kidneys was demonstrated by immunohistochemistry, immunofluorescence, and electron microscopy, while single-cell RNA sequencing of human kidneys showed expression of the cognate gene, SPOCK2, exclusively in podocytes. In vitro, testican-2 increased glomerular endothelial tube formation and motility, raising the possibility that its secretion has a functional role within the glomerulus. Taken together, our findings identify testican-2 as a podocyte-derived biomarker of kidney health and prognosis.

Molecular Characterisation of a Rare Reassortant Porcine-Like G5P[6] Rotavirus Strain Detected in an Unvaccinated Child in Kasama, Zambia

A human-porcine reassortant strain, RVA/Human-wt/ZMB/UFS-NGS-MRC-DPRU4723/2014/G5P[6], was identified in a sample collected in 2014 from an unvaccinated 12 month old male hospitalised for gastroenteritis in Zambia. We sequenced and characterised the complete genome of this strain which presented the constellation: G5-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The genotype A8 is often observed in porcine strains. Phylogenetic analyses showed that VP6, VP7, NSP2, NSP4, and NSP5 genes were closely related to cognate gene sequences of porcine strains (e.g., RVA/Pig-wt/CHN/DZ-2/2013/G5P[X] for VP7) from the NCBI database, while VP1, VP3, VP4, and NSP3 were closely related to porcine-like human strains (e.g., RVA/Human-wt/CHN/E931/2008/G4P[6] for VP1, and VP3). On the other hand, the origin of the VP2 was not clear from our analyses, as it was not only close to both porcine (e.g., RVA/Pig-tc/CHN/SWU-1C/2018/G9P[13]) and porcine-like human strains (e.g., RVA/Human-wt/LKA/R1207/2009/G4P[6]) but also to three human strains (e.g., RVA/Human-wt/USA/1476/1974/G1P[8]). The VP7 gene was located in lineage II that comprised only porcine strains, which suggests the occurrence of independent porcine-to-human reassortment events. The study strain may have collectively been derived through interspecies transmission, or through reassortment event(s) involving strains of porcine and porcine-like human origin. The results of this study underline the importance of whole-genome characterisation of rotavirus strains and provide insights into interspecies transmissions from porcine to humans.

Targeted intragenic demethylation initiates chromatin rewiring for gene activation

Aberrant DNA methylation in the region surrounding the transcription start site is a hallmark of gene silencing in cancer. Currently approved demethylating agents lack specificity and exhibit high toxicity. Herein we show, using the p16 gene as an example, that targeted demethylation of the promoter-exon 1-intron 1 (PrExI) region initiates an epigenetic wave of local chromatin remodeling and distal long-range interactions, culminating in gene-locus specific activation. Through development of CRISPR-DiR (DNMT1-interacting RNA), in which ad hoc edited guides block methyltransferase activity in a locus-specific fashion, we demonstrate that demethylation is coupled to epigenetic and topological changes. These results suggest the existence of a specialized “demethylation firing center (DFC)” which can be switched on by an adaptable and selective RNA-mediated approach for locus-specific transcriptional activation.One Sentence SummaryLocus demethylation via CRISPR-DiR reshapes chromatin structure and specifically reactivates its cognate gene.

Oxidative Catabolism of (+)-Pinoresinol Is Initiated by an Unusual Flavocytochrome Encoded by Translationally Coupled Genes within a Cluster of (+)-Pinoresinol-Coinduced Genes in Pseudomonas sp. Strain SG-MS2

ABSTRACT Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a Km of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes. IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β′ linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.