Targeting of proteins to chromatin in Drosophila melanogaster

Dosage compensation of sex chromosomes in Drosophila melanogaster is an excellent model system to study various aspects of targeting of protein factors to chromatin. Dosage compensation prevents male lethality by up regulating transcription from the single male X chromosome in the ~2 fold range to match the two active X chromosomes in females [reviewed in e.g. (Ferrari et al., 2014; Kuroda et al., 2016; Samata and Akhtar, 2018)]. This up regulation is facilitated by the male specific lethal (MSL) dosage compensation complex (DCC). The DCC binds selectively to ~300 high affinity sites (HAS) on the X chromosome, containing a low complexity GAGA rich sequence motif, the MSL recognition element (MRE) (Alekseyenko et al., 2008; Straub et al., 2008). However, the DCC neglects thousands of other similar sequences in the genome outside of HAS. The DNA binding subunit MSL2 alone can enrich X chromosomal MREs in vitro, although MSL2 misses most MREs within HAS (Villa et al., 2016). The Chromatin Linked Adaptor for MSL Proteins (CLAMP) binds thousands of MREs genome wide and contributes to DCC targeting to HAS (Kaye et al., 2018; Soruco et al., 2013). The role of CLAMP in facilitating MSL2 targeting to HAS was investigated by several approaches. Monitoring MSL2 chromatin binding in vivo by chromatin immunoprecipitation with high throughput sequencing (ChIP seq) showed the requirement of CLAMP for HAS targeting. Next, the interplay between CLAMP and MSL2 in genome wide in vitro DNA binding was studied by DNA immunoprecipitation with high throughput sequencing (DIP seq) (Gossett and Lieb, 2008; Liu et al., 2005; Villa et al., 2016). The data revealed mutual recruitment of both factors to each other’s binding sites and cooperative binding to novel sites. This DNA binding cooperativity extended each other’s binding repertoire to facilitate robust binding of MREs located within HAS, although increased binding to other non functional sites was observed. Both factors interacted directly with each other in co IP experiments, providing an explanation for cooperative DNA binding. Whether CLAMP and MSL2 are required for keeping HAS nucleosome free was studied by assay for transposase accessibly chromatin with high throughput sequencing (ATAC seq) (Buenrostro et al., 2013; Buenrostro et al., 2015). Both factors cooperate to stabilize each other’s binding and to compete with nucleosome positioning at HAS. After successful binding of the DCC to HAS, it interacts with neighboring target genes, which are marked by trimethylation of histone H3K36 (H3K36me3). There, the DCC catalyzes acetylation of H4K16 (H4K16ac) to boost transcription (Akhtar and Becker, 2000; Gelbart et al., 2009; Larschan et al., 2007; Prestel et al., 2010). The DCC employs the chromosome 3D organization, which seems to be invariant between males and females, to transfer from HAS to active genes (Ramirez et al., 2015; Ulianov et al., 2016). The contribution of HAS to the chromosome interaction network was studied by using different chromosome conformation capture techniques. Hi C analysis on sex sorted embryos showed that, H4K16ac and H3K36me3 correlate well with the active compartments (Sexton et al., 2012). Interestingly, compartment switching on the X chromosome between males and females was correlated with H4K16ac and therefore attributed to dosage compensation. The involvement of the Pioneering sites on the X (PionX), a special sub-class of HAS, in chromosome architecture was studied by high resolution 4C seq in male and female cells. Chromosomal segments containing PionX made frequent contact with many loci within the active compartment and even looped over large domains of the inactive compartment (Ghavi-Helm et al., 2014). These long range interactions between PionX with other PionX/HAS were more robust in males compared to females, indicating that the dosage compensation machinery reinforced them. Moreover, de novo induction of DCC assembly in female cells showed that the DCC uses long range interaction within the active compartment to transfer from PionX to target genes marked by H3K36me3 for up regulation of transcription. The chromosomal kinase JIL 1, which catalyzes phosphorylation of histone H3S10, localizes also to actively transcribed genes marked by H3K36me3 and is two fold enriched on the male X chromosome (Jin et al., 2000; Regnard et al., 2011; Wang et al., 2001). JIL 1 is implicated in maintaining overall chromosome organization and preventing the spreading of heterochromatin into the euchromatic part of the X chromosome in both sexes (Cai et al., 2014; Ebert et al., 2004; Jin et al., 1999). Furthermore, JIL 1 localizes to the non LTR retrotransposon arrays of the telomeres to positively regulate their expression (Andreyeva et al., 2005; Silva-Sousa and Casacuberta, 2013; Silva-Sousa et al., 2012). The role of JIL 1 in regulating gene expression was studied using various methods. JIL 1 formed a stable complex with the novel PWWP domain containing protein, JIL 1 Anchoring and Stabilizing Protein (JASPer). The JIL 1 JASPer (JJ) complex specifically enriched H3K36me3 modified nucleosomes in vitro via JASPer’s PWWP domain from a nucleosome library containing 115 different nucleosome types. Consistently, ChIP seq experiments showed that the JJ complex localizes to H3K36me3 chromatin at active gene bodies and at telomeric transposons in vivo. As previously described, the JJ complex is also enriched on the male X chromosome relative to autosomes. Loss of JIL 1 resulted in loss of JASPer enrichment, a small increase in H3K9me2 and a decrease in H4K16ac on the X chromosome shown by spike in ChIP seq. Gene expression analysis by RNA seq showed that the JJ complex positively regulates expression of genes, in particular of genes from the male X chromosome, and of telomeric transposons. Furthermore, the JJ complex associated with the Set1/COMPASS complex and with other remodelling complexes as shown by co IP coupled to mass spectrometry analysis

Serdemetan Antagonizes the Mdm2-HIF1α Axis Leading to Decreased Levels of Glycolytic Enzymes

Serdemetan (JNJ-26854165), an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1), were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises the possibility of side effects associated with therapeutically targeting Mdm2

Matrix Gla Protein Reinforces Angiogenic Resolution

Matrix Gla Protein (MGP) is an ECM molecule commonly associated with dysfunctions of large blood vessels such as arteriosclerosis and atherosclerosis. However, the exact role of MGP in the microvasculature is not clear. Utilizing a mouse MGP knockout model we found that MGP suppresses angiogenic sprouting from mouse aorta restricts microvascular density in cardiac and skeletal muscle, and is an endogenous inhibitor of tumor angiogenesis. Similarly, morpholino based knockdown of MGP in zebrafish embryos caused a progressive loss of luminal structures in intersegmental vessels, a phenotype reminiscent of Dll4/Notch inhibition. Accordingly, MGP suppressed Notch-dependent Hes-1 promoter activity and expression of Jagged1 mRNA relative to Dll4 mRNA. However, inhibition of BMP but not Notch or VEGF signaling reversed the excessive angiogenic sprouting phenotype of MGP knockout aortic rings suggesting that MGP may normally suppress angiogenic sprouting by blocking BMP signaling. Collectively, these results suggest that MGP is a multi-functional inhibitor of normal and abnormal angiogenesis that may function by coordinating with both Notch and BMP signaling pathways

Delayed wound repair in sepsis is associated with reduced local pro-inflammatory cytokine expression

Sepsis is one of the main causes for morbidity and mortality in hospitalized patients. Moreover, sepsis associated complications involving impaired wound healing are common. Septic patients often require surgical interventions that in-turn may lead to further complications caused by impaired wound healing. We established a mouse model to the study delayed wound healing during sepsis distant to the septic focus point. For this reason cecal ligation and puncture (CLP) was combined with the creation of a superficial wound on the mouse ear. Control animals received the same procedure without CPL. Epithelialization was measured every second day by direct microscopic visualization up to complete closure of the wound. As interplay of TNF-α, TGF-β, matrix metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) is important in wound healing in general, TNF-α, TGF-β, MMP7, and TIMP1 were assessed immunohistochemical in samples of wounded ears harvested on days 2, 6, 10 and 16 after wounding. After induction of sepsis, animals showed a significant delay in wound epithelialization from day 2 to 12 compared to control animals. Complete wound healing was attained after mean 12.2± standard deviation (SD) 3.0 days in septic animals compared to 8.7± SD 1.7 days in the control group. Septic animals showed a significant reduction in local pro-inflammatory cytokine level of TNF-α on day 2 and day 6 as well as a reduced expression of TGF-β on day 2 in wounds. A significant lower expression of MMP7 as well as TIMP1 was also observed on day 2 after wounding. The induction of sepsis impairs wound healing distant to the septic focus point. We could demonstrate that expression of important cytokines for wound repair is deregulated after induction of sepsis. Thus restoring normal cytokine response locally in wounds could be a good strategy to enhance wound repair in sepsis

A Comparison of Resveratrol and Other Polyphenolic Compounds on Notch Activation and Endothelial Cell Activity

Resveratrol is a polyphenolic compound produced by plants which makes its way into the human diet through plant-based foods. It has been shown to provide many health benefits, helping to ward of age-related diseases and promoting cardiovascular health. Additionally, resveratrol is a potent activator of the Notch signaling pathway. While resveratrol receives the most attention as a polyphenolic nutraceutical, other compounds with similar structures may be more potent regulators of specific cellular processes. Here, we compare resveratrol, apigenin, chrysin, genistein, luteolin, myricetin, piceatannol, pterostilbene, and quercetin for their ability to regulate Notch signaling. In addition, we compare the ability of these polyphenolic compounds to regulate endothelial cell viability, proliferation, and migration. Out of these compounds we found that resveratrol is the best activator of Notch signaling, however, other similar compounds are also capable of stimulating Notch. We also discovered that several of these polyphenols were able to inhibit endothelial cell proliferation. Finally, we found that many of these polyphenols are potent inhibitors of endothelial migration during wound healing assays. These findings provide the first side-by-side comparison of the regulation of Notch signaling, and endothelial cell proliferation and migration, by nine polyphenolic compounds

Lipocalin-7 Is a Matricellular Regulator of Angiogenesis

Matricellular proteins are extracellular regulators of cellular adhesion, signaling and performing a variety of physiological behaviors such as proliferation, migration and differentiation. Within vascular microenvironments, matricellular proteins exert both positive and negative regulatory cues to vascular endothelium. The relative balance of these matricellular cues is believed to be critical for vascular homeostasis, angiogenesis activation or angiogenesis resolution. However, our knowledge of matricellular proteins within vascular microenvironments and the mechanisms by which these proteins impact vascular function remain largely undefined. The matricellular protein lipocalin-7 (LCN7) is found throughout vascular microenvironments, and circumstantial evidence suggests that LCN7 may be an important regulator of angiogenesis. Therefore, we hypothesized that LCN7 may be an important regulator of vascular function.To test this hypothesis, we examined the effect of LCN7 overexpression, recombinant protein and gene knockdown in a series of in vitro and in vivo models of angiogenesis. We found that overexpression of LCN7 in MB114 and SVEC murine endothelial cell lines or administration of highly purified recombinant LCN7 protein increased endothelial cell invasion. Similarly, LCN7 increased angiogenic sprouting from quiescent endothelial cell monolayers and ex vivo aortic rings. Moreover, LCN7 increased endothelial cell sensitivity to TGF-β but did not affect sensitivity to other pro-angiogenic growth factors including bFGF and VEGF. Finally, morpholino based knockdown of LCN7 in zebrafish embryos specifically inhibited angiogenic sprouting but did not affect vasculogenesis within injected embryos.No functional analysis has previously been performed to elucidate the function of LCN7 in vascular or other cellular processes. Collectively, our results show for the first time that LCN7 is an important pro-angiogenic matricellular protein of vascular microenvironments

MAGP2 Controls Notch via Interactions with RGD Binding Integrins: Identification of a Novel ECM-Integrin-Notch Signaling Axis

Canonical Notch signaling involves Notch receptor activation via interaction with cell surface bound Notch ligand. Recent findings also indicate that Notch signaling may be modulated by cross-talk with other signaling mechanisms. The ECM protein MAGP2 was previously shown to regulate Notch in a cell type dependent manner, although the molecular details of this interaction have not been dissected. Here, we report that MAGP2 cell type specific control of Notch is independent of individual Notch receptor-ligand combinations but dependent on interaction with RGD binding integrins. Overexpressed MAGP2 was found to suppress transcriptional activity from the Notch responsive Hes1 promoter activity in endothelial cells, while overexpression of a RGD→RGE MAGP2 mutant increased Notch signaling in the same cell type. This effect was not unique to MAGP2 since the RGD domain of the ECM protein EGFL7 was also found to be an important modulator of Hes1 promoter activity. Independently of MAGP2 or EGFL7, inhibition of RGD-binding integrins with soluble RGD peptides also increased accumulation of active N1ICD fragments and Notch responsive promoter activity independently of changes in Notch1, Jag1, or Dll4 expression. Finally, β1 or β3 integrin blocking antibodies also enhanced Notch signaling. Collectively, these results answer the question of how MAGP2 controls cell type dependent Notch signaling, but more importantly uncover a new mechanism to understand how extracellular matrices and cellular environments impact Notch signaling

Ornithine Decarboxylase mRNA is Stabilized in an mTORC1-dependent Manner in Ras-transformed Cells

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development