RNA-binding proteins (RBPs) are crucial in modulating RNA metabolism in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Although previous studies on the conservation of ...RBP targets have been carried out in lower eukaryotes such as yeast, relatively little is known about the extent of conservation of the binding sites of RBPs across mammalian species.
In this study, we employ CLIP-seq datasets for 60 human RBPs and demonstrate that most binding sites for a third of these RBPs are conserved in at least 50% of the studied vertebrate species. Across the studied RBPs, binding sites were found to exhibit a median conservation of 58%, ~ 20% higher than random genomic locations, suggesting a significantly higher preservation of RBP-RNA interaction networks across vertebrates. RBP binding sites were highly conserved across primates with weak conservation profiles in birds and fishes. We also note that phylogenetic relationship between members of an RBP family does not explain the extent of conservation of their binding sites across species. Multivariate analysis to uncover features contributing to differences in the extents of conservation of binding sites across RBPs revealed RBP expression level and number of post-transcriptional targets to be the most prominent factors. Examination of the location of binding sites at the gene level confirmed that binding sites occurring on the 3' region of a gene are highly conserved across species with 90% of the RBPs exhibiting a significantly higher conservation of binding sites in 3' regions of a gene than those occurring in the 5'. Gene set enrichment analysis on the extent of conservation of binding sites to identify significantly associated human phenotypes revealed an enrichment for multiple developmental abnormalities.
Our results suggest that binding sites of human RBPs are highly conserved across primates with weak conservation profiles in lower vertebrates and evolutionary relationship between members of an RBP family does not explain the extent of conservation of their binding sites. Expression level and number of targets of an RBP are important factors contributing to the differences in the extent of conservation of binding sites. RBP binding sites on 3' ends of a gene are the most conserved across species. Phenotypic analysis on the extent of conservation of binding sites revealed the importance of lineage-specific developmental events in post-transcriptional regulatory network evolution.
Abuse, neglect, and other forms of early life stress (ELS) significantly increase risk for psychiatric disorders including depression. In this study, we show that ELS in a postnatal sensitive period ...increases sensitivity to adult stress in female mice, consistent with our earlier findings in male mice. We used RNA-sequencing in the ventral tegmental area, nucleus accumbens, and prefrontal cortex of male and female mice to show that adult stress is distinctly represented in the brain's transcriptome depending on ELS history. We identify: 1) biological pathways disrupted after ELS and associated with increased behavioral stress sensitivity, 2) putative transcriptional regulators of the effect of ELS on adult stress response, and 3) subsets of primed genes specifically associated with latent behavioral changes. We also provide transcriptomic evidence that ELS increases sensitivity to future stress through enhancement of known programs of cortical plasticity.
Vulnerability to relapse during periods of attempted abstinence from cocaine use is hypothesized to result from the rewiring of brain reward circuitries, particularly ventral tegmental area (VTA) ...dopamine neurons. How cocaine exposures act on midbrain dopamine neurons to precipitate addiction-relevant changes in gene expression is unclear. We found that histone H3 glutamine 5 dopaminylation (H3Q5dop) plays a critical role in cocaine-induced transcriptional plasticity in the midbrain. Rats undergoing withdrawal from cocaine showed an accumulation of H3Q5dop in the VTA. By reducing H3Q5dop in the VTA during withdrawal, we reversed cocaine-mediated gene expression changes, attenuated dopamine release in the nucleus accumbens, and reduced cocaine-seeking behavior. These findings establish a neurotransmission-independent role for nuclear dopamine in relapse-related transcriptional plasticity in the VTA.
Schizophrenia (SZ) is a psychiatric disorder with complex genetic risk dictated by interactions between hundreds of risk variants. Epigenetic factors, such as histone posttranslational modifications ...(PTMs), have been shown to play critical roles in many neurodevelopmental processes, and when perturbed may also contribute to the precipitation of disease. Here, we apply an unbiased proteomics approach to evaluate combinatorial histone PTMs in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons from individuals with SZ. We observe hyperacetylation of H2A.Z and H4 in neurons derived from SZ cases, results that were confirmed in postmortem human brain. We demonstrate that the bromodomain and extraterminal (BET) protein, BRD4, is a bona fide 'reader' of H2A.Z acetylation, and further provide evidence that BET family protein inhibition ameliorates transcriptional abnormalities in patient-derived neurons. Thus, treatments aimed at alleviating BET protein interactions with hyperacetylated histones may aid in the prevention or treatment of SZ.
Animals susceptible to chronic social defeat stress (CSDS) exhibit depression-related behaviors, with aberrant transcription across several limbic brain regions, most notably in the nucleus accumbens ...(NAc). Early life stress (ELS) promotes susceptibility to CSDS in adulthood, but associated enduring changes in transcriptional control mechanisms in the NAc have not yet been investigated. In this study, we examined long-lasting changes to histone modifications in the NAc of male and female mice exposed to ELS. Dimethylation of lysine 79 of histone H3 (H3K79me2) and the enzymes (DOT1L and KDM2B) that control this modification are enriched in D2-type medium spiny neurons and are shown to be crucial for the expression of ELS-induced stress susceptibility. We mapped the site-specific regulation of this histone mark genome wide to reveal the transcriptional networks it modulates. Finally, systemic delivery of a small molecule inhibitor of DOT1L reversed ELS-induced behavioral deficits, indicating the clinical relevance of this epigenetic mechanism.
Abstract
Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs. However, the ...underlying mechanisms remain unclear. Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion. By applying conditional deletion of
Bmal1
in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications. Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis. Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-of-day effect on axon growth potential. Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration.
The regeneration of peripheral nerves is guided by regeneration tracks formed through an interplay of many cell types, but the underlying signaling pathways remain unclear. Here, we demonstrate that ...macrophages are mobilized ahead of Schwann cells in the nerve bridge after transection injury to participate in building regeneration tracks. This requires the function of guidance receptor Plexin-B2, which is robustly up-regulated in infiltrating macrophages in injured nerves. Conditional deletion of Plexin-B2 in myeloid lineage resulted in not only macrophage misalignment but also matrix disarray and Schwann cell disorganization, leading to misguided axons and delayed functional recovery. Plexin-B2 is not required for macrophage recruitment or activation but enables macrophages to steer clear of colliding axons, in particular the growth cones at the tip of regenerating axons, leading to parallel alignment postcollision. Together, our studies unveil a novel reparative function of macrophages and the importance of Plexin-B2-mediated collision-dependent contact avoidance between macrophages and regenerating axons in forming regeneration tracks during peripheral nerve regeneration.
Iron deficiency (ID) during the fetal-neonatal period results in long-term neurodevelopmental impairments associated with pervasive hippocampal gene dysregulation. Prenatal choline supplementation ...partially normalizes these effects, suggesting an interaction between iron and choline in hippocampal transcriptome regulation. To understand the regulatory mechanisms, we investigated epigenetic marks of genes with altered chromatin accessibility (ATAC-seq) or poised to be repressed (H3K9me3 ChIP-seq) in iron-repleted adult rats having experienced fetal-neonatal ID exposure with or without prenatal choline supplementation.
Fetal-neonatal ID was induced by limiting maternal iron intake from gestational day (G) 2 through postnatal day (P) 7. Half of the pregnant dams were given supplemental choline (5.0 g/kg) from G11-18. This resulted in 4 groups at P65 (Iron-sufficient IS, Formerly Iron-deficient FID, IS with choline ISch, and FID with choline FIDch). Hippocampi were collected from P65 iron-repleted male offspring and analyzed for chromatin accessibility and H3K9me3 enrichment. 22% and 24% of differentially transcribed genes in FID- and FIDch-groups, respectively, exhibited significant differences in chromatin accessibility, whereas 1.7% and 13% exhibited significant differences in H3K9me3 enrichment. These changes mapped onto gene networks regulating synaptic plasticity, neuroinflammation, and reward circuits. Motif analysis of differentially modified genomic sites revealed significantly stronger choline effects than early-life ID and identified multiple epigenetically modified transcription factor binding sites.
This study reveals genome-wide, stable epigenetic changes and epigenetically modifiable gene networks associated with specific chromatin marks in the hippocampus, and lays a foundation to further elucidate iron-dependent epigenetic mechanisms that underlie the long-term effects of fetal-neonatal ID, choline, and their interactions.
Ketamine has rapid and sustained antidepressant effects in patients with treatment-resistant depression (TRD). However, the underlying mechanisms of action are not well understood. There is ...increasing evidence that TRD is associated with a pro-inflammatory state and that ketamine may inhibit inflammatory processes. We thus investigated whole blood transcriptional profiles related to TRD and gene expression changes associated with treatment response to ketamine. Whole blood was collected at baseline (21 healthy controls HC, 26 patients with TRD) and then again in patients with TRD 24 hours following a single intravenous infusion of ketamine (0.5 mg/kg). We performed RNA-sequencing and analyzed (a) baseline transcriptional profiles between patients with TRD and HC, (b) responders vs. non-responders before ketamine treatment, and (c) gene expression signatures associated with clinical improvement. At baseline, patients with TRD compared to HC showed a gene expression signature indicative of interferon signaling pathway activation. Prior to ketamine administration, the metabotropic glutamate receptor gene GRM2 and the ionotropic glutamate receptor gene GRIN2D were upregulated in responders compared to non-responders. Response to ketamine was associated with a distinct transcriptional signature, however, we did not observe gene expression changes indicative of an anti-inflammatory effect. Future studies are needed to determine the role of the peripheral immune system in the antidepressant effect of ketamine.
Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor ...patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.
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