Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-[3H]lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size. The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting. The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury. This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction. In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled. Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively. Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion. High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled. Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet. Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I. For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes. The enzyme may be localized in the globin domain in erythrocytes and maintains that region in a hyperacetylated state which results in an altered linker histone binding reflected in a change in the reactivity of the usually inaccessible H3 cysteine 110.
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PMID:Histone H3 thiol reactivity and acetyltransferases in chicken erythrocyte nuclei. 317 Jun 3

Only a small fraction of the adult chicken erythrocyte histones is involved in dynamic acetylation. We have reported previously that the rapidly acetylated and deacetylated H4 histones are primarily associated with the transcriptionally active DNA-enriched chromatin fragments that remain attached to the residual nuclear material following micrococcal nuclease digestion and chromatin solubilization. Furthermore, this nuclear fraction contained most of the histone deacetylase activity. In this study we show that the bulk of the nuclear histone acetyltransferase activity is located with the insoluble residual nuclear material. We demonstrate that in vitro the enzymes associated with the residual nuclear material catalyze reversible acetylation when the endogenous histones of the nuclear skeleton-bound chromatin fragments are used as substrate. Nuclear matrices isolated from adult chicken immature erythrocyte and trout liver nuclei had 60-76% of the nuclear histone acetyltransferase activity. Procedures that solubilized the internal nuclear matrix also resulted in the release of the enzyme from the nuclear matrix. Together, our observations suggest that histone acetyltransferase and deacetylase are associated with the internal nuclear matrix, and one of the functions of these enzymes may be to mediate a dynamic attachment between transcriptionally active chromatin and the nuclear matrix.
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PMID:Histone acetyltransferase is associated with the nuclear matrix. 807 41

We have examined salt-soluble chromatin released by micrococcal nuclease from a 15-day-old chicken embryo erythrocyte nuclei for histone acetyltransferase (HAT) activities. This chromatin is enriched in transcriptionally active sequences from within the active beta-globin locus and contains elevated levels of acetylated core histones. HAT activities present in this fraction target histones H4, H3, and H2A when the chromatin itself is used as the substrate. In gel HAT activity assay demonstrates that the salt-soluble chromatin fraction contains four acetyltransferase molecules distinguished by their different molecular masses (47, 33, 32, and 28 kDa). Further separation of the chromatin by centrifugation through sucrose gradients shows that the acetyltransferases segregate into chromatin-bound and chromatin-free populations. The 32- and 28-kDa HATs are associated with chromatin, whereas the 47- and 33-kDa HAT molecules are not. The chromatin-bound HAT activities predominantly target H4 to give the diacetyl and triacetyl species; some acetylation of H2A can also be seen. Our results suggest that the chromatin-associated acetyltransferases have a role in gene regulation.
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PMID:Multiple histone acetyltransferases are associated with a chicken erythrocyte chromatin fraction enriched in active genes. 1089 66

In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.
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PMID:Dual roles of p300 in chromatin assembly and transcriptional activation in cooperation with nucleosome assembly protein 1 in vitro. 1194 Jun 55

The estrogen receptor plays an important role in breast cancer progression. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), also called modulator of nongenomic activity of estrogen receptor (MNAR), a novel coactivator of estrogen receptor, modulates estrogen receptor transactivation functions. The mechanisms by which PELP1 modulates estrogen receptor genomic functions is not known. Here, using biochemical and scanning confocal microscopic analysis, we have demonstrated nuclear localization and functional implications of PELP1. Subnuclear fractionation showed PELP1 association with chromatin and nuclear matrix fractions. Ligand stimulation promoted recruitment of PELP1 to 17beta-estradiol responsive promoters, its colocalization with acetylated H3, and increased PELP1-associated histone acetyltransferase enzymatic activity. Far Western analysis revealed that PELP1 interacts with histone 1 and 3, with more preference toward histone 1. Using deletion analysis, we have identified the PELP1 COOH-terminal region as the histone 1 binding site. The PELP1 mutant lacking histone 1-binding domain acts as a dominant-negative and blocks estrogen receptor alpha-mediated transcription. Chromatin immunoprecipitation analysis showed a cyclic association and dissociation of PELP1 with the promoter, with recruitment of histone 1 and PELP1 occurring in opposite phases. PELP1 overexpression increased the micrococcal nuclease sensitivity of estrogen response element-containing nucleosomes. Our results provide novel insights about the transcription regulation of PELP1 and suggest that PELP1 participates in chromatin remodeling activity via displacement of histone 1 in cancer cells.
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PMID:Potential role of a novel transcriptional coactivator PELP1 in histone H1 displacement in cancer cells. 1537 49

STAT6 is a critical regulator of transcription for interleukin-4 (IL-4)-induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. Coactivator proteins CBP/p300 have been implicated in regulation of transcription in all STATs. CBP is also required for STAT6-mediated gene activation, but the underlying molecular mechanisms are still elusive. In this study we investigated the mechanisms by which STAT6 recruits CBP and chromatin-modifying activities to the promoter. Our results indicate that while STAT1-interacted directly with CBP, the interaction between STAT6 and CBP was found to be mediated through p100 protein, a coactivator protein that has previously been shown to stimulate the transcription of IL-4-induced genes. The staphylococcal nuclease-like (SN)-domains of p100 directly interacted with amino acids 1099-1758 of CBP, while p100 did not associate with SRC-1, another coactivator of STAT6. p100 was found to recruit histone acetyltransferase (HAT) activity to STAT6 in vivo. Chromatin immunoprecipitation studies demonstrated that p100 increases the STAT6-p100-CBP ternary complex formation in the human Igepsilon promoter. p100 also increased the amount of acetylated histone H4 at the Igepsilon promoter, and siRNAs directed against p100 effectively inhibited Igepsilon reporter gene expression. Our results suggest that p100 has an important role in the assembly of STAT6 transcriptosome, and that p100 stimulates IL-4-dependent transcription by mediating interaction between STAT6 and CBP and recruiting chromatin modifying activities to STAT6-responsive promoters.
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PMID:The transcriptional co-activator protein p100 recruits histone acetyltransferase activity to STAT6 and mediates interaction between the CREB-binding protein and STAT6. 1569 2

We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how to measure the degree of chromatin assembly in the absence and presence of histone H1 using topological analysis and how to perform micrococcal nuclease digestion to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further, we describe the use of sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).
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PMID:Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context. 1730 35

In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control), the elp3 (one of histone acetyltransferase genes) deletion mutant, and the hos2 (one of histone deactylase genes) deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants.
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PMID:Comparative studies of genome-wide maps of nucleosomes between deletion mutants of elp3 and hos2 genes of Saccharomyces cerevisiae. 2129 80

Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. We show here that a histone H2A variant, Htz1 (H2A.Z), in nucleosomes has a positive function in promoting efficient NER in yeast. Htz1 inherently enhances the occupancy of the histone acetyltransferase Gcn5 on chromatin to promote histone H3 acetylation after UV irradiation. Consequently, this results in an increased binding of a NER protein, Rad14, to damaged DNA. Cells without Htz1 show increased UV sensitivity and defective removal of UV-induced DNA damage in the Htz1-bearing nucleosomes at the repressed MFA2 promoter, but not in the HMRa locus where Htz1 is normally absent. Thus, the effect of Htz1 on NER is specifically relevant to its presence in chromatin within a damaged region. The chromatin accessibility to micrococcal nuclease in the MFA2 promoter is unaffected by HTZ1 deletion. Acetylation on previously identified lysines of Htz1 plays little role in NER or cell survival after UV. In summary, we have identified a novel aspect of chromatin that regulates efficient NER, and we provide a model for how Htz1 influences NER in Htz1 nucleosomes.
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PMID:Histone variant Htz1 promotes histone H3 acetylation to enhance nucleotide excision repair in Htz1 nucleosomes. 2392 26

Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein implicated in a variety of cellular processes. In the present study, we identified Tudor-SN as a novel regulator in cell cycle. Tudor-SN was abundant in proliferating cells whereas barely expressed in terminally differentiated cells. Functional analysis indicated that ectopic overexpression of Tudor-SN promoted the G1/S transition, whereas knockdown of Tudor-SN caused G1 arrest. Moreover, the live-cell time-lapse experiment demonstrated that the cell cycle of MEF(-/-) (knock-out of Tudor-SN in mouse embryonic fibroblasts) was prolonged compared with wild-type MEF(+/+). We noticed that Tudor-SN was constantly expressed in every cell cycle phase, but was highly phosphorylated in the G1/S border. Further study revealed that Tudor-SN was a potential substrate of Cdk2/4/6, supportively, we found the physical interaction of endogenous Tudor-SN with Cdk4/6 in G1 and the G1/S border, and with Cdk2 in the G1/S border and S phase. In addition, roscovitine (Cdk1/2/5 inhibitor) or CINK4 (Cdk4/6 inhibitor) could inhibit the phosphorylation of Tudor-SN, whereas ectopic overexpression of Cdk2/4/6 increased the Tudor-SN phosphorylation. The underlying molecular mechanisms indicated that Tudor-SN could physically interact with E2F-1 in vivo, and could enhance the physical association of E2F-1 with GCN5 (a cofactor of E2F-1, which possesses histone acetyltransferase activity), and promote the binding ability of E2F-1 to the promoter region of its target genes CYCLIN A and E2F-1, and as a result, facilitate the gene transcriptional activation. Taken together, Tudor-SN is identified as a novel co-activator of E2F-1, which could facilitate E2F-1-mediated gene transcriptional activation of target genes, which play essential roles in G1/S transition.
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PMID:Tudor staphylococcal nuclease (Tudor-SN), a novel regulator facilitating G1/S phase transition, acting as a co-activator of E2F-1 in cell cycle regulation. 2562 88


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