A duplicated copy of id2b is an unusual sex-determining candidate gene on the Y chromosome of arapaima (Arapaima gigas)

Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGFβ signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.

EGF stimulates human trophoblast cell invasion by downregulating ID3-mediated KISS1 expression

Background During pregnancy, trophoblast cell invasion needs to be finely controlled. Aberrant trophoblast cell invasion is associated with placental diseases. Epidermal growth factor (EGF) and its receptor, EGFR, are expressed in trophoblast cells. Although the pro-invasive effect of EGF on trophoblast cells has been reported, the underlying mechanism remains largely unknown. Results In the present study, we conducted an RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to EGF and identified KISS1 as a target gene of EGF. The human KISS1 gene encodes kisspeptin, also known as metastin, which can suppress tumor metastasis. Our results showed that EGF treatment downregulated KISS1 expression and secretion by activating the EGFR-mediated PI3K/AKT signaling pathway. In addition, the expression of inhibitor of DNA-binding protein 3 (ID3) was downregulated by EGF and that was required for the EGF-suppressed KISS1 expression. Functionally, transwell invasion assays demonstrated that EGF stimulated human trophoblast cell invasion by downregulating KISS1 expression. Preeclampsia (PE) is a placental disease characterized by insufficient trophoblast cell invasion. Our clinical results revealed that serum levels of EGF were downregulated while serum and placental levels of KISS1 were upregulated in PE patients. Conclusions This study demonstrates that downregulation of EGF can lead to poor trophoblast cell invasion by increasing KISS1 expression which subsequently contributes to the pathogenesis of PE.

Inhibitor of DNA binding 2 (Id2) mediates microtubule polymerization in the brain by regulating αK40 acetylation of α-tubulin

Acetylation of α-tubulin lysine 40 (αK40) contributes to microtubule (MT) stability and is essential for neuronal development and function, whereas excessive αK40 deacetylation is observed in neurodegenerative disorders including Alzheimer’s disease (AD). Here we identified inhibitor of DNA binding 2 (Id2) as a novel MT-binding partner that interacts with α-tubulin and enhances αK40 acetylation, leading to MT polymerization in the neurons. Commensurate with our finding that the low levels of Id2 expression along with a reduced αK40 acetylation in the postmortem human AD patient and 5X-FAD, AD model mice brain, Id2 upregulation in the hippocampus of 5X-FAD, which exhibit high levels of Sirt2 expression, increased αK40 acetylation and reconstitutes axon growth. Hence our study suggests that Id2 is critical for maintaining MT stability during neural development and the potential of Id2 to counteract pathogenic Sirt2 activity in AD.

Targeted Disruption of the Inhibitor of DNA Binding 4 (Id4) Gene Alters Photic Entrainment of the Circadian Clock

Inhibitor of DNA binding (Id) genes comprise a family of four helix–loop–helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4−/− mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4−/− SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4−/− mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4−/− photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4−/− retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2−/− mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4−/− SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.

Discovery of novel ID2 antagonists from pharmacophore-based virtual screening as potential therapeutics for glioma

Glioma, especially the most aggressive type glioblastoma multiforme, is one of the central nervous system malignant cancer with a poor prognosis. Traditional treatments are mainly surgery combined with radiotherapy and chemotherapy, which is still not satisfactory. Therefore, it is of great clinical significance to find new therapeutic agents. Served as an inhibitor of differentiation, protein ID2 (inhibitor of DNA binding 2) plays an important role in neurogenesis, neovascularization and malignant development of gliomas. It has been shown that ID2 affects the malignant progression of gliomas through different mechanisms. In this study, a pharmacophore-based virtual screening was carried out and 16 hit compounds were purchased for pharmacological evaluations on their ID2 inhibitory activities. Based on the cytotoxicity of these small-molecule compounds, two compounds were shown to effectively inhibit the viability of glioma cells in the low micromolar range. Among them, AK-778-XXMU was chosen for further study due to its better solubility in water. A SPR assay proved the high affinity between AK-778-XXMU and ID2 protein with the KD value as 129 nM. The plausible binding mode in the biding site of ID2 was studied by molecular docking. Subsequently, the cancer-suppressing potency of the compound was characterized both in vitro and in vivo. The data demonstrated that compound AK-778-XXMU is a potent ID2 antagonist which has the potential to be developed as a therapeutic agent against glioma.

Inhibitor of DNA-binding family regulates the prognosis of ovarian cancer

Aim: The mechanistic role of inhibitor of DNA binding or differentiation (ID) family in ovarian cancer (OC) has remained unclear. Materials & methods: We used the Oncomine, GEPIA, Kaplan–Meier Plotter, cBioPortal, SurvExpress, PROGgene V2, TIMER, and FunRich to evaluate the prognostic value of IDs in patients with OC. Results: the mRNA transcripts of all IDs were markedly downregulated in OC compared with normal tissue. The prognostic value of IDs was also explored within the subtypes, pathological stages, clinical stages and TP53 mutational status. The group with low-risk IDs showed relatively good overall survival (OS) compared with the high-risk group. Conclusion: ID1/3/4 may be exploited as promising prognostic biomarkers and therapeutic targets in OC patients.

Altered Responsiveness to TGFβ and BMP and Increased CD45+ Cell Presence in Mitral Valves Are Unique Features of Ischemic Mitral Regurgitation

Objective: Ischemic mitral regurgitation (IMR) often develops after an ischemic event, which results in distortion of the valvulo-ventricular complex and incomplete mitral valve (MV) leaflet coaptation. After left ventricular ischemic events, only some patients develop IMR. The susceptibility of the MV to remodel may influence whether IMR develops. We hypothesized that impaired signaling response in MV cells may contribute to IMR development by inducing maladaptive tissue remodeling. Approach and Results: Sheep (n=14) were subjected to ligation of the circumflex coronary artery to induce myocardial infarction. IMR was reported by echocardiography. MV leaflets and MV interstitial cells (MVICs) were collected at baseline (control, n=10), 4 and 8 weeks post-myocardial infarction. RNA sequencing highlighted differences in TGFβ (transforming growth factor beta) signaling between MV with/without IMR. SMAD6/7 and ID2 (inhibitor of DNA binding 2) were the highest increased TGFβ-signaling genes associated with IMR. MVICs from myocardial infarction sheep were less responsive to BMP (bone morphogenic protein) 4 pro-osteogenic stimulation (ID2, OPN [osteopontin], and OC [osteocalcin] mRNA) than control. MVICs from IMR sheep had a diminished COL (collagen) 1A1 mRNA response to TGFβ1 and enhanced prochondrogenic RUNX2 (runt-related transcription factor 2) and SOX9 mRNA response to BMP4 versus non-IMR MVICs. Baseline CD45 expression was detectable only in IMR MVICs. Upon TGFβ1 stimulation, CD45 expression was detected in all groups. Immunostaining confirmed increased presence of CD45+ cells in IMR MV interstitium. Conclusions: MVs from sheep with IMR had an altered TGFβ/BMP response, associated with increased CD45+ cell presence within the tissue interstitium. Pharmacological strategies aimed to modulate TGFβ/BMP signaling after myocardial infarction may protect from pathological MV remodeling leading to IMR.