Digestion by micrococcal nuclease of mouse submandibular-salivary-gland chromatin.
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ABSTRACT:
SUBMITTER: Smith GJ
PROVIDER: S-EPMC1161800 | biostudies-other | 1980 May
REPOSITORIES: biostudies-other
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Project description:Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.
| S-EPMC3012088 | biostudies-literature
Project description:PurposeSalivary fluid formation is primarily driven by Ca(2+)-activated, apical efflux of chloride into the lumen of the salivary acinus. The anoctamin1 protein is an anion channel with properties resembling the endogenous calcium-activated chloride channels. In order to better understand the role of anoctamin proteins in salivary exocrine secretion, the expression of the ten members of the anoctamin gene family in the mouse submandibular gland was studied.MethodsTotal RNA extracted from mouse submandibular salivary glands was reverse transcribed using primer pairs to amplify the full-length coding regions of each anoctamin gene and was subcloned into plasmid vectors for DNA sequencing. Alternative splice variants were also screened by polymerase chain reaction using primer pairs that amplified six overlapping regions of the complementary DNA of each anoctamin gene, spanning multiple exons.ResultsMultiple anoctamin transcripts were found in the mouse submandibular salivary gland, including full-length transcripts of anoctamin1, anoctamin3, anoctamin4, anoctamin5, anoctamin6, anoctamin9, and anoctamin10. Exon-skipping splicing in the N-terminal exons of the anoctamins1, anoctamin5, and anoctamin6 genes resulted in multiple alternative splice variants. No expression of anoctamin2, anoctamin7, or anoctamin8 was found.ConclusionsThe predominant anoctamin transcript expressed in the mouse submandibular gland is anoctamin1ac. The chloride channel protein produced by anoctamin1ac is likely responsible for the Ca(2+)-activated chloride efflux, which is the rate-limiting step in salivary exocrine secretion.
| S-EPMC4415004 | biostudies-literature
Project description:Activation of an apical Ca(2+)-dependent Cl(-) channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca(2+)-gated Cl(-) currents generated by Tmem16A and Best2, members from two distinct families of Ca(2+)-activated Cl(-) channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca(2+)-activated Cl(-) currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca(2+)-activated Cl(-) current found in native salivary acinar cells. Best2(-/-) and Tmem16A(-/-) mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca(2+)-activated Cl(-) current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A(-/-) mice lacked a Ca(2+)-activated Cl(-) current and a Ca(2+)-mobilizing agonist failed to stimulate Cl(-) efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2(-/-) mice. Our results demonstrate that both Tmem16A and Best2 generate Ca(2+)-activated Cl(-) current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca(2+)-activated Cl(-) channel complex that is essential for saliva production by the submandibular gland.
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Project description:[32P]Pi was administered to rats (5mCi/rat) 2h before the isolation of liver nuclei. The isolated nuclei were subjected to mild micrococcal-nuclease digestion for 2.5, 5 and 10 min at 37 degrees C, and the mononucleosomal fraction was subsequently isolated by sucrose-density-gradient centrifugation. The specific radioactivity of 32P-labelled mononucleosomal fractions decreased with increased digestion times. A phosphorylated chromosomal protein, B2 (Mr 68000, pI6.5-8.2), was demonstrated immunologically in the mononucleosomal fraction by using an antibody specific to this electrophoretically purified phosphoprotein. The incorporation of 32P into this phosphoprotein, previously shown to be mainly through covalent linkage, was revealed by antibody precipitation followed by gel electrophoresis. The rate of release of acid-soluble nucleotides by micrococcal-nuclease digestion of liver nuclei from partially hepatectomized rats 16 h after operation was strikingly higher than that for sham-operated controls. After partial hepatectomy, an increase in 32P incorporation into phosphoprotein in the monomer fractions specifically precipitated by this antibody was also found. This suggests that the phosphorylated non-histone chromatin protein B2 is preferentially associated with the transcriptionally active chromatin.
| S-EPMC1153657 | biostudies-other
Project description:BackgroundOrgan replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. However, the generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown.MethodsIsolated human submandibular gland stem/progenitor cells (hSMGepiS/PCs) were characterized and three-dimensionally (3D) cultured to generate organoids and further induced by fibroblast growth factor 10 (FGF10) in vitro. The induced spheres alone or in combination with embryonic day 12.5 (E12.5) mouse salivary gland mesenchyme were transplanted into the renal capsules of nude mice to assess their development in vivo. Immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, calcium release analysis, western blotting, hematoxylin-eosin staining, Alcian blue-periodic acid-Schiff staining, and Masson's trichrome staining were performed to assess the structure and function of generated tissues in vitro and in vivo.ResultsThe isolated hSMGepiS/PCs could be long-term cultured with a stable genome. The organoids treated with FGF10 [FGF10 (+) group] exhibited higher expression of salivary gland-specific markers; showed spatial arrangement of AQP5+, K19+, and SMA+ cells; and were more sensitive to the stimulation by neurotransmitters than untreated organoids [FGF10 (-) group]. After heterotopic transplantation, the induced cell spheres combined with mouse embryonic salivary gland mesenchyme showed characteristics of mature salivary glands, including a natural morphology and saliva secretion.ConclusionFGF10 promoted the development of the hSMGepiS/PC-derived salivary gland organoids by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. Moreover, the hSMGepiS/PCs responded to the niche in mouse embryonic mesenchyme and further differentiated into salivary gland tissues with mature characteristics. Our study provides a foundation for the regenerative therapy of salivary gland diseases.
| S-EPMC7083056 | biostudies-literature
Project description:Previous studies demonstrated that salivary gland morphogenesis and differentiation are enhanced by modification of fibrin hydrogels chemically conjugated to Laminin-1 peptides. Specifically, Laminin-1 peptides (A99: CGGALRGDN-amide and YIGSR: CGGADPGYIGSRGAA-amide) chemically conjugated to fibrin promoted formation of newly organized salivary epithelium both in vitro (e.g., using organoids) and in vivo (e.g., in a wounded mouse model). While these studies were successful, the model's usefulness for inducing regenerative patterns after radiation therapy remains unknown. Therefore, the goal of the current study was to determine whether transdermal injection with the Laminin-1 peptides A99 and YIGSR chemically conjugated to fibrin hydrogels promotes tissue regeneration in irradiated salivary glands. Results indicate that A99 and YIGSR chemically conjugated to fibrin hydrogels promote formation of functional salivary tissue when transdermally injected to irradiated salivary glands. In contrast, when left untreated, irradiated salivary glands display a loss in structure and functionality. Together, these studies indicate that fibrin hydrogel-based implantable scaffolds containing Laminin-1 peptides promote secretory function of irradiated salivary glands.
| S-EPMC8498954 | biostudies-literature
Project description:UnlabelledA 15 year old boy presented with swelling in the submandibular region. X ray of the part showed faint radio opaque shadow. A provisional diagnosis of sialadenitis with sialiolithiasis was made. Excised mass was reported histopathologically as plexiform neurofibroma of submandibular salivary gland. Plexiform neurofibroma of the salivary gland is a rare benign tumour, often present in the parotid gland. It is very rare in submandibular salivary gland. It is a slow growing, locally infiltrating tumour.Electronic supplementary materialThe online version of this article (doi:10.1007/s12262-010-0174-5) contains supplementary material, which is available to authorized users.
| S-EPMC3087058 | biostudies-literature
Project description:Histone post-translational modifications (PTMs) such as acetylation and methylation are known to affect chromatin higher order structures. Primary targets of these modifications include basic residues present at N-terminus tail region of core histones. Four histone acetyltransferase (HAT) genes have been identified in trypanosomatids. HAT1, HAT3 and HAT4 of Leishmania donovani have been partially characterized. However, there is no report about HAT2 of Leishmania donovani. Lysine residues present on the N-terminal tail of Leishmania donovani histone H4 are conserved in other trypanosomatids and humans. PTMs of lysines modulate various functions at chromatin level. The four histone acetyltransferases encoded in Leishmania genome were over-expressed to analyse their functional activity. All four HATs were found actively acetylating core histones H3/H4. Similar to L. donovani HAT3 and HAT4, HAT2 was found to be a member of MYST family protein and have SAS2 type domain. Over-expression of HAT2 significantly increases acetylation of H4K4. To analyse the effect of HAT2 over-expression on chromatin accessibility, micrococcal nuclease digestion assay was performed. MNase digestion resulted in a higher proportion of the mononucleosomes and dinucleosomes in HAT2 over-expressing cells as compared to WT L. donovani cells. Acetylation of lysine-4 neutralizes the amino terminal region of histone H4. This weakens its interaction with neighbouring nucleosomes and the linker DNA. HAT2 over-expression in L. donovani resulted in highly accessible chromatin suggesting chromatin decondensation. HAT2 may have an important role to play in global regulation of transcription in L. donovani. Better understanding of these epigenetic determinants of parasite would help in designing novel therapeutic strategies.
| S-EPMC5423686 | biostudies-literature
Project description:The nuclear lamina has been shown to interact with the genome through lamina-associated domains (LADs). LADs have been identified by DamID, a proximity labeling assay, and more recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs form megabase-size domains at the nuclear periphery, they are gene-poor and mostly heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely bath sonication or digestion with micrococcal nuclease (MNase), leads to the discovery of common but also distinct sets of lamin-interacting domains, or LiDs. Using ChIP-seq, we show the existence of lamin A/C (LMNA) LiDs with distinct gene contents, histone composition enrichment and relationships to lamin B1-interacting domains. The extent of genome coverage of lamin A/C (LMNA) LiDs in sonicated or MNase-digested chromatin is similar (∼730 megabases); however over half of these domains are uniquely detected in sonicated or MNase-digested chromatin. Sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, while MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. LMNB1 LiDs are gene-poor and show no or little enrichment in these marks. Comparison of published LMNB1 DamID LADs with LMNB1 and LMNA LiDs identified here by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin characteristics, and which are not associated with LMNB1. The differential genomic and epigenetic properties of lamin-interacting domains reflect the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible regions presumably localized in the nuclear interior.
| S-EPMC4615303 | biostudies-literature
Project description:ObjectivesThe Salivary Gland Committee of the American Academy of Otolaryngology-Head and Neck Surgery seeks to standardize terminology and technique for ultrasonograpy used in the evaluation and treatment of salivary gland disorders.MethodsDevelopment of expert opinion obtained through interaction with international practitioners representing multiple specialties. This committee work includes a comprehensive literature review with presentation of case examples to propose a standardized protocol for the language used in ultrasound salivary gland assessment.ResultsA multiple segment proposal is initiated with this focus on the submandibular gland. We provide a concise rationale for recommended descriptive language highlighted by a more extensive supplement that includes an extensive literature review with additional case examples.ConclusionRecommendations are provided to improve consistency both in performing and reporting submandibular gland ultrasound.
| S-EPMC10866606 | biostudies-literature