High Mobility Group Nucleosomal Binding Research Articles

Pan-cancer Multi-omics Analysis Reveals HMGN1 as a Potential Prognostic and Immune Infiltration-associated Biomarker.

The High Mobility Group Nucleosomal Binding Domain 1 Gene (HMGN1) is crucial for epigenetic regulation. However, the specific function of HMGN1 in cancer development is unclear. Raw data on HMGN1 expression were procured from Genotype-Tissue Expression (GTEx), the University of Alabama- Birmingham CANcer data analysis Portal (UALCAN), and The Cancer Genome Atlas (TCGA). Thereafter, the pan-cancer analysis was implemented to understand the HMGN1 expression patterns, prognostic value, and immunological features. Furthermore, the Gene Set Enrichment Analysis (GSEA) was executed via R language. In addition, the relationship between HMGN1 and the sensitivity of antitumor drugs was also determined. Finally, real-time PCR (RT-PCR) experiments were carried out. Pan-cancer analysis revealed that HMGN1 was upregulated in several solid tumors and was associated with pathological staging and poor prognosis. In addition, HMGN1 was found to be involved in regulating the tumor microenvironment. The GSEA enrichment analysis indicated that HMGN1 assisted in the regulation of oncogenic processes, especially metabolic and immune pathways. Furthermore, HMGN1 expression was linked to microsatellite instability (MSI) and tumor mutational burden (TMB) across diverse tumor types. RT-PCR assays indicated that HMGN1 was overexpressed in the gastric and breast cancer cell lines and tissues. This study highlighted the potential of HMGN1 as a biomarker for pan- - cancer analysis.

WTAP dysregulation-mediated HMGN3-m6A modification inhibited trophoblast invasion in early-onset preeclampsia.

Early-onset preeclampsia (ePE) originates from abnormal implantation and placentation that involves trophoblast invasion, but its pathophysiology is not entirely understood. N6-methyladenosine (m6A) regulators mediate the progression of various cancers. The invasiveness of trophoblast cells is similar to that of tumor cells. However, little is known regarding the potential role of m6A modification in ePE and the underlying mechanism. This study aimed to explore the m6A level in placental tissue samples collected from ePE patients and to investigate whether m6A modification was an essential part of PE pathogenesis. The m6A level in placental tissue samples of 80 PE participants was examined. MeRIP-microarray, RNA-Seq, luciferase reporter assay, and RNA immunoprecipitation chip (RIP) assay were performed. The m6A level in the ePE group was significantly reduced compared with the control group. Wilms' tumor 1-associating protein (WTAP) regulated trophoblast cell migration and invasion. Mechanistically, the high mobility group nucleosomal binding domain 3 (HMGN3) gene was a target gene of WTAP in trophoblast (p < .05). WTAP enhanced the stability of HMGN3 mRNA through binding with its 3'-UTR m6A site(+485A, +522A). HMGN3 was recognized by m6A recognition protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which was inhibited when knocking down WTAP. Both m6A and WTAP levels were downregulated in ePE. The m6A modification mediated by WTAP/IGF2BP1/HMGN3 axis might contribute to abnormal trophoblast invasion. Our work provided a foundation for further exploration of RNA epigenetic regulatory patterns in ePE, and indicated a new treatment strategy for ePE.

The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin-induced autophagy in macrophages by affecting Ulk1 transcription.

Pyocyanin (PYO) is a major virulence factor secreted by Pseudomonas aeruginosa, and autophagy is a crucial homeostatic mechanism for the interaction between the pathogens and the host. It remains unknown whether PYO leads to autophagy in macrophages by regulating histone acetylation. The high mobility group nucleosomal binding domain 2 (HMGN2) has been reported to regulate the PYO‐induced autophagy and oxidative stress in the epithelial cells; however, the underlying molecular mechanism has not been fully elucidated. In this study, PYO was found to induce autophagy in macrophages, and the mechanism might be correlated with the up‐regulation of HMGN2 acetylation (HMGN2ac) and the down‐regulation of H3K27 acetylation (H3K27ac) by modulation of the activities of acetyltransferases and deacetylases. Moreover, we further demonstrated that the up‐regulated HMGN2ac enhances its recruitment to the Ulk1 promoter, while the down‐regulation of H3K27ac reduces its recruitment to the Ulk1 promoter, thereby promoting or inhibiting the transcription of Ulk1. In conclusion, HMGN2ac and H3K27ac play regulatory roles in the PYO‐induced autophagy in macrophages.

Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells.

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.

High mobility group nucleosomal binding 2 reduces integrin α5/β1‐mediated adhesion of Klebsiella pneumoniae on human pulmonary epithelial cells via nuclear factor I

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/β1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/β1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/β1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/β1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/β1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.

Transcriptomic analysis of peripheral blood mononuclear cells in head and neck squamous cell carcinoma patients.

To investigate the gene expression profile of peripheral blood mononuclear cells (PBMCs) from head and neck squamous cell carcinoma (HNSCC), including oral cancer (OC) and oropharyngeal cancer (OPC) patients, and compare them with healthy controls (HC). Transcriptomic analysis of PBMCs was performed by RNA-sequencing. The upregulated candidate genes were selected for validation by quantitative real-time polymerase chain reaction (qPCR). In addition, related plasma protein levels were determined by enzyme-linked immunosorbent assay (ELISA). Three significantly upregulated genes, including high mobility group nucleosomal binding domain 2 (HMGN2), folate receptor gamma (FOLR3), and amphiregulin (AREG), were selected. In the first cohort, the results showed that only HMGN2 expression was significantly increased in OC patients. In the larger sample size, the overall results demonstrated that HMGN2 expression had a tendency to increase in both OC and OPC patients compared with HC. Interestingly, the plasma HMGN2 (HMG-17) protein level exhibited the same trend as that observed at the transcriptional level. HMGN2 expression and plasma HMG-17 (HMGN2 protein) were increased in both cancer patients compared with HC. This gene may be important for further functional studies in the PBMCs of HNSCC patients.

High-mobility group nucleosomal binding domain 2 protects against microcephaly by maintaining global chromatin accessibility during corticogenesis

The surface area of the human cerebral cortex undergoes dramatic expansion during late fetal development, leading to cortical folding, an evolutionary feature not present in rodents. Microcephaly is a neurodevelopmental disorder defined by an abnormally small brain, and many gene mutations have been found to be associated with primary microcephaly. However, mouse models generated by ablating primary microcephaly-associated genes often fail to recapitulate the severe loss of cortical surface area observed in individuals with this pathology. Here, we show that a mouse model with deficient expression of high-mobility group nucleosomal binding domain 2 (HMGN2) manifests microcephaly with reduced cortical surface area and almost normal radial corticogenesis, with a pattern of incomplete penetrance. We revealed that altered cleavage plane and mitotic delay of ventricular radial glia may explain the rising ratio of intermediate progenitor cells to radial glia and the displacement of neural progenitor cells in microcephalic mutant mice. These led to decreased self-renewal of the radial glia and reduction in lateral expansion. Furthermore, we found that HMGN2 protected corticogenesis by maintaining global chromatin accessibility mainly at promoter regions, thereby ensuring the correct regulation of the transcriptome. Our findings underscore the importance of the regulation of chromatin structure in cortical development and highlight a mouse model with critical insights into the etiology of microcephaly.

Construction and dissection of the ceRNA‑ceRNA network reveals critical modules in depression.

The prevalence of mental health disorders such as depression is high. Depression is a multifactorial disorder and its underlying mechanisms remain unclear. Competing endogenous RNA (ceRNA) regulation has been reported to serve important roles in human disease. In the present study, ceRNA networks for depression and the corresponding normal physiological states were constructed. Further analysis of the ceRNA networks revealed that ceRNA regulation may be important for depression. Hub ceRNAs including high mobility group nucleosomal binding domain 3, peroxisome proliferator-activated receptor-γ coactivator 1β and leukemia inhibitory factor receptor-α were associated with depression. A common core ceRNA network was identified by comparison analysis. Functional analysis suggested that these ceRNAs may be implicated in depression. Differential expression analysis revealed that ceRNAs in the obtained ceRNA interaction networks were significantly enriched with significantly differentially expressed genes. A total of 8 key functional modules for depression were identified, and small target molecules were screened. ceRNA protocadherin-α subfamily C2 in module 1 and ceRNA Cyclin-dependent kinase 6 in module 3 were reported to be implicated in the occurrence and development of depressive disorders. Thus, the present analysis may provide insight into the pathogenesis of depression and improve its treatment.

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells

The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling.

Knockdown of HMGN2 increases the internalization of Klebsiellapneumoniae by respiratory epithelial cells through the regulation of α5β1 integrin expression.

Integrin receptors, a large family of adhesion receptors, are involved in the attachment of Klebsiella pneumoniae to respiratory epithelial cells, and subsequently cause the internalization of K. pneumoniae by host cells. Although a number of molecules have been reported to regulate the expression and activity of integrin receptors in respiratory epithelial cells, the specific underlying molecular mechanisms remain largely unknown. High mobility group nucleosomal binding domain 2 (HMGN2), a non-histone nuclear protein, is present in eukaryotic cells as a ubiquitous nuclear protein. Our previous studies have demonstrated that HMGN2 affects chromatin function and modulates the expression of antibacterial peptide in A549 cells exposed to lipopolysaccharide, which indicates the critical role of HMGN2 in innate immune responses. In addition, our cDNA microarray analysis suggested that HMGN2 knockdown induced the enhanced expression of α5β1 integrin in A549 cells. Therefore, we hypothesized that intercellular HMGN2 may mediate the internalization of K. pneumoniae by altering the expression of α5β1 integrin. Using the A549 cell line, we demonstrated that HMGN2 knockdown induced the increased expression of α5β1 integrin on cell membranes, which resulted in a significant increase in K. pneumoniae internalization. Further results revealed that HMGN2 silencing induced the expression of talin and the activation of α5β1 integrin, which led to actin polymerization following the phosphorylation of FAK and Src. This study suggests a possible therapeutic application for bacterial internalization by targeting HMGN2 in order to treat K. pneumoniae infection.

High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) SUMOylation by the SUMO E3 Ligase PIAS1 Decreases the Binding Affinity to Nucleosome Core Particles

High mobility group nucleosomal binding domain 2 (HMGN2) is a small and unique non-histone protein that has many functions in a variety of cellular processes, including regulation of chromatin structure, transcription, and DNA repair. In addition, it may have other roles in antimicrobial activity, cell homing, and regulating cytokine release. Although the biochemical properties of HMGN2 protein are regulated by acetylation and phosphorylation, it is not yet known whether HMGN2 activity can also be regulated by SUMOylation. In this study, we demonstrated for the first time that HMGN2 is modified by covalent attachment of small ubiquitin-related modifier 1 (SUMO1) by pro-inflammatory signal and identified the major SUMOylated lysine residues that localize to the HMGN2 nucleosome-binding domain at Lys-17 and Lys-35. SENP1 can deSUMOylate SUMOylated HMGN2, and PIAS1 is the E3 ligase responsible for SUMOylation of HMGN2. Finally, using SUMO1-conjugated HMGN2 purified from a basal SUMOylation system in Escherichia coli, we demonstrated that SUMOylated HMGN2 has decreased the binding affinity to nucleosome core particles in comparison to unSUMOylated HMGN2. These observations potentially provide new perspectives for understanding the functions of HMGN2 in inflammatory reaction.

Environmental manipulations generate bidirectional shifts in both behavior and gene regulation in a crossbred mouse model of extremes in trait anxiety

Although gene-environment interactions are known to significantly influence psychopathology-related disease states, only few animal models cover both the genetic background and environmental manipulations. Therefore, we have taken advantage of the bidirectionally inbred high (HAB) and low (LAB) anxiety-related behavior mouse lines to generate HAB × LAB F1 hybrids that intrinsically carry both lines’ genetic characteristics, and subsequently raised them in three different environments—standard, enriched (EE) and chronic mild stress (CMS). Assessing genetic correlates of trait anxiety, we focused on two genes already known to play a role in HAB vs. LAB mice, corticotropin releasing hormone receptor type 1 (Crhr1) and high mobility group nucleosomal binding domain 3 (Hmgn3). While EE F1 mice showed decreased anxiety-related and increased explorative behaviors compared to controls, CMS sparked effects in the opposite direction. However, environmental treatments affected the expression of the two genes in distinct ways. Thus, while expression ratios of Hmgn3 between the HAB- and LAB-specific alleles remained equal, total expression resembled the one observed in HAB vs. LAB mice, i.e., decreased after EE and increased after CMS treatment. On the other hand, while total expression of Crhr1 remained unchanged between the groups, the relative expression of HAB- and LAB-specific alleles showed a clear effect following the environmental modifications. Thus, the environmentally driven bidirectional shift of trait anxiety in this F1 model strongly correlated with Hmgn3 expression, irrespective of allele-specific expression patterns that retained the proportions of basic differential HAB vs. LAB expression, making this gene a match for environment-induced modifications. An involvement of Crhr1 in the bidirectional behavioral shift could, however, rather be due to different effects of the HAB- and LAB-specific alleles described here. Both candidate genes therefore deserve attention in the complex regulation of anxiety-related phenotypes including environment-mediated effects.

Nucleosome-binding protein HMGN2 exhibits antitumor activity in oral squamous cell carcinoma

Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) serve as effectors in the antitumor response. High mobility group nucleosomal binding domain 2 (HMGN2) is a candidate effector molecule involved in CTL and NK cell function. In the current study, recombinant human HMGN2 was isolated and purified from transformed Escherichia coli. Tca8113 cells, an oral squamous cell carcinoma line, were treated with a variety of HMGN2 protein concentrations and cell growth was analyzed. HMGN2 significantly inhibited the growth of Tca8113 cells and was predicted to arrest cells in the S phase. Moreover, HMGN2 treatment increased the apoptosis rate of Tca8113 cells. Western blotting indicated the upregulation of p53 and Bax proteins, whereas Bcl-2 was significantly downregulated. In addition, caspase-3 was found to be activated. Furthermore, the HMGN2 protein may suppress the growth of Tca8113 cells in vivo. The results of the current study indicated that the HMGN2 protein may inhibit the growth of oral squamous cell carcinoma and HMGN2 may represent an antitumor effector molecule of CTL or NK cells.

Genome-wide association study identifies HMGN3 locus for spine bone size variation in Chinese.

Bone size (BS) is one of the major risk factors for osteoporotic fractures. BS variation is genetically determined to a substantial degree with heritability over 50%, but specific genes underlying variation of BS are still largely unknown. To identify specific genes for BS in Chinese, initial genome-wide association scan (GWAS) study and follow-up replication study were performed. In initial GWAS study, a group of 12 contiguous single-nucleotide polymorphism (SNP)s, which span a region of ~25 kb and locate at the upstream of HMGN3 gene (high-mobility group nucleosomal binding domain 3), achieved moderate association signals for spine BS, with P values ranging from 6.2E-05 to 1.8E-06. In the follow-up replication study, eight of the 12 SNPs were detected suggestive replicate associations with BS in 1,728 unrelated female Caucasians, which have well-known differences from Chinese in ethnic genetic background. The SNPs in the region of HMGN3 gene formed a tightly combined haplotype block in both Chinese and Caucasians. The results suggest that the genomic region containing HMGN3 gene may be associated with spine BS in Chinese.

Profiling Trait Anxiety: Transcriptome Analysis Reveals Cathepsin B (Ctsb) as a Novel Candidate Gene for Emotionality in Mice

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait “anxiety”. We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior.We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4).Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females.Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

High Mobility Group Nucleosomal Binding Domain 2 Protein Protects Bladder Epithelial Cells from Klebsiella pneumoniae Invasion

Due to the predominance of multiple-antibiotic-resistant Klebsiella pneumoniae strains, the search for new approaches for the prevention of K. pneumoniae infections is now under intensive investigation. The objective of the present study was to investigate the effects of high mobility group nucleosomal binding domain 2 (HMGN2) protein, which acts on the bladder epithelial cells T 24, on the invasion of K. pneumoniae 03183 and explore its possible mechanisms. Pretreatment with HMGN2 significantly reduced K. pneumoniae 03183 uptake by T 24 cells. In T 24 cells, there were no detectable cytotoxic effects of HMGN2 at any concentration between 32 and 256 µg/ml after 2 h incubation. HMGN2 exhibited no appreciable antibacterial activity against K. pneumoniae 03183. Fluorescence microscopy and flow cytometry analysis revealed that HMGN2 blocked K. pneumoniae 03183-induced actin polymerization. K. pneumoniae 03183-induced phosphorylation of extracellular signal-regulated kinase (ERK) and cofilin were prevented by pretreatment with HMGN2. These results indicated that pretreatment with HMGN2 inhibited cofilin phosphorylation and then induced actin disruption which may block ERK phosphorylation. These changes led to inhibition of K. pneumoniae 03183 invasion of T 24 bladder epithelial cells.

Downregulation of the nucleosome-binding protein 1 (NSBP1) gene can inhibit the in vitro and in vivo proliferation of prostate cancer cells

This study is to construct a lentiviral vector harbouring an RNA interference (RNAi) sequence that targets the gene encoding the human high-mobility group nucleosomal binding protein 1 (NSBP1); to study its role in inducing G(2)/M phase arrest and apoptosis in prostate cancer (PCa) DU145 cells; and to assess the effect of its knockdown on cell proliferation in vitro and in vivo. RNAi was applied to knock down NSBP1 expression in the PCa cell line DU145 by lentiviral plasmids producing an NSBP1 small hairpin RNA. After NSBP1 knockdown in DU145 cells, the growth rate of cells was analyzed by MTT, and G(2)/M cell cycle arrest and apoptosis were assessed using a FACScalibur flow cytometer. Tumour growth was assessed in nude mice. The mRNA and protein expression levels of NSBP1, cyclin B1 and Bcl-2 were analysed in vitro and in vivo by reverse-transcriptase polymerase chain reaction and Western blotting. Knockdown of NSBP1 resulted in a 22.6% decrease in the growth rate of cells compared with the PscNC lentivirus control cells at 96 h, decreased tumour growth in nude mice, and the induction of G2/M cell cycle arrest (8.78%) and apoptosis (2.19-fold). Consistent with the cell cycle arrest and apoptosis, the mRNA and protein expression levels of cyclin B1 and Bcl-2 were decreased. In conclusion, knockdown of NSBP1 causes a statistically significant inhibition of the in vitro and in vivo growth of the PCa cell line DU145. Growth suppression is at least partially due to NSBP1 knockdown-induced G2/M cell cycle arrest and apoptosis. The present data provide the evidence that the NSBP1 knockdown-induced G2/M phase arrest and apoptosis may result from negative regulation of cyclin B1 and Bcl-2 by NSBP1, with the resulting reduced expression of these proteins.

Gene expression during minor genome activation in preimplantation bovine development

The main goal of this study was to identify mRNA transcripts whose content increases during bovine minor embryonic genome activation. We compared the gene expression profile of the bovine 4-cell-stage embryo and MII oocyte using the technique of suppression subtractive hybridization. Differentially expressed amplicons were subcloned, and 60 of them were sequenced. The resulting DNA sequences were compared with GenBank databases using BLAST search. The expression of five differentially expressed genes with an apparent function in cell cycle progression, chromatin remodeling, and splicing or translation initiation was further characterized by a real-time RT-PCR. Centromere protein F, 350/400 ka (CENPF), and splicing factor arginine/serine-rich 3 (SRFS3) show an increase in mRNA content during the 2- to 4-cell and late 8-cell stages. For the high mobility group nucleosomal binding domain 2 (HMGN2), the level of mRNA increases in 2- to 4-cell and morula embryos. The transcription of splicing factor SRFS3 is α-amanitin sensitive both during 4-cell and late 8-cell stages. The transcription of CENPF and HMGN2 is α-amanitin sensitive only at late 8-cell stage and morula, respectively. SRFS3 represents the first described gene with an important function in preimplantation development, which is also expressed during bovine minor genome activation, and it is α-amanitin sensitive during this period. All described genes can play an important role in the preimplantation development of bovine embryos.

Alpha-Helical domain is essential for antimicrobial activity of high mobility group nucleosomal binding domain 2 (HMGN2)1

To examine the antimicrobial spectrum and functional structure of high mobility group nucleosomal binding domain 2 (HMGN2). OMIGA protein structure software was used to analyze the two-dimensional structure of HMGN2. Synthetic short peptides were generated for studying the relationship between function and structure. Prokaryotic expression vectors were constructed for the holo-HMGN2 and its helical domain. Their E coli-based products were also prepared for antimicrobial testing. The antimicrobial assay included minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration. OMIGA protein structure software analysis revealed a transmembrane alpha-helical structure (the putative antimicrobial domain) located from position 18 to 48 of the HMGN2 protein sequence. The antimicrobial assay showed that the MIC of the recombinant holo-HMGN2 against E coli ML-35p (an ampicillin-resistance strain), Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231 were 12.5, 25, and 100 mg/L, respectively. Against the same microorganisms, the MIC of the synthetic HMGN2 alpha-helical domain were 12.5, 25, and 100 mg/L, respectively, that is, the same as with the recombinant form of HMGN2. In contrast, recombinant holo-HMGN2 was inactive against Staphylococcus aureus ATCC 25923. The synthetic N-terminal and C-terminal fragments of HMGN2 had no antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 or C albicans ATCC 10231. HMGN2 showed potent antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 and, to some extent, against C albicans ATCC 10231, but was inactive against S aureus ATCC 25923 in these assay systems. Itos alpha-helical structure may be essential for the antimicrobial activity of HMGN2.

Activation of the nuclear factor kappa B pathway following ischemia-reperfusion of the murine testis.

Ischemia-reperfusion (IR) of the testis results in testicular oxidative stress and germ cell-specific apoptosis. Nuclear factor kappa B (NF-kappaB) is a nuclear transcription factor involved in the control of a number of cellular processes, and its activation is part of the cellular stress response to a variety of factors including cytokine stimulation, irradiation, and IR. The present study investigates NF-kappaB activation after IR of the murine testis and potential downstream target genes of that activation. Mice were subjected to a period of testicular ischemia followed by 0-4 hours of reperfusion. Activation of NF-kappaB was assessed by 1) Western blot analysis of the NF-kappaB inhibitory protein, IkappaBalpha; 2) immunohistochemistry for IkappaBalpha; and 3) TranSignal NF-kappaB target gene array (107 genes) analysis. Results demonstrate that IkappaBalpha is phosphorylated on serine 32 reaching a peak by 2 hours after IR of the testis. A decrease in total IkappaBalpha was also noted at 2 hours after IR, consistent with the rapid degradation of the phosphorylated protein. Phosphorylation and degradation of IkappaBalpha is indicative of NF-kappaB activation. Immunolocalization revealed IkappaBalpha specifically in Sertoli cells of the murine testis. Results of the TranSignal target gene array revealed that the expression of 9 genes was consistently changed 2 hours after IR of the testis, 3 of which increased in expression and 6 of which were down-regulated. Most notably, high-mobility group nucleosomal binding domain 1 increased in expression while platelet-derived growth factor B and Wilms tumor homolog decreased. These results suggest that testicular IR releases the suppression of NF-kappaB by IkappaBalpha in Sertoli cells. Activation of the NF-kappaB pathway in the testis resulted in an alteration of expression of potential NF-kappaB target genes, some increased while others decreased. The specific roles of these genes in the testicular response to IR remains to be determined.