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Functional specificity of CpG DNA-binding CXXC domains in mixed lineage leukemia.
The MLL CXXC domain binds nonmethylated CpG-containing DNA and is essential for the oncogenic properties of MLL fusion proteins. To determine potential functional promiscuity of similar DNA binding domains, we replaced the MLL CXXC domain in the context of the leukemogenic MLL-AF9 fusion with CXXC domains from DNMT1, CGBP (CFP1), and MBD1, or with a methyl-CpG-binding domain (MBD) from MBD1. MLL(DNMT1 CXXC)-AF9 shows robust in vitro colony forming activity and in vivo leukemogenesis, similar to MLL-AF9. However, colony forming ability and leukemogenicity are abrogated in MLL-AF9 containing either the CGBP or MBD1 CXXC domains or the MBD1 MBD domain. Direct comparison of in vitro DNA binding affinity of the isolated CXXC or MBD domains demonstrated that MLL, DNMT1, and CGBP CXXC domains could each bind to unmethylated DNA but with differing affinity. In contrast, the isolated MBD1 CXXC and MBD1 MBD domains were unable to bind to the same DNA. However, all substituted domains still allowed targeting of the MLL fusions to the functionally important Hoxa9 locus in primary bone marrow progenitor cells. In addition to DNA binding activity, it was critical that specific CpG residues in the Hoxa9 locus were protected from methylation for leukemia development. This ultimately prevented histone 3 lysine 9 trimethylation (H3K9me3) of the locus and enabled Hoxa9 expression. These were properties shared by MLL and DNMT1 CXXC domains but not by CGBP CXXC or the other swapped fusions tested. We demonstrate that similar CXXC domains can be mechanistically distinguished by specificity of CpG nucleotides preferentially protected from DNA methylation.
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17-10086
Nombre del producto:

EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit
Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells.
Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of "ectopic" H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents "leakage" of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3.
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07-473
Nombre del producto:

Anti-trimethyl-Histone H3 (Lys4) Antibody
The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology, and the metabolome of fibroblasts.
Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC domain-containing histone demethylase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 consists of a CxxC domain, a PHD domain, and an Fbox domain. By purifying the JmjC and the PHD domain of Fbxl10 and using different approaches we were able to characterize the properties of these domains in vitro. Our results suggest that Fbxl10 is rather a H3K4me3 than a H3K36me2 histone demethylase. The PHD domain exerts a dual function in binding H3K4me3 and H3K36me2 and exhibiting E3 ubiquitin ligase activity. We generated mouse embryonic fibroblasts stably overexpressing Fbxl10. These cells reveal an increase in cell size but no changes in proliferation, mitosis, or apoptosis. Using a microarray approach we were able to identify potentially new target genes for Fbxl10 including chemokines, the noncoding RNA Xist, and proteins involved in metabolic processes. Additionally, we found that Fbxl10 is recruited to the promoters of Ccl7, Xist, Crabp2, and RipK3. Promoter occupancy by Fbxl10 was accompanied by reduced levels of H3K4me3 but unchanged levels of H3K36me2. Furthermore, knockdown of Fbxl10 using small interfering RNA approaches showed inverse regulation of Fbxl10 target genes. In summary, our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control of fibroblasts.
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Múltiplo
Nombre del producto:

Múltiplo
A single point mutation in the LN domain of LAMA2 causes muscular dystrophy and peripheral amyelination.
Mutations in the gene encoding the basal lamina (BL) component laminin alpha2 (LAMA2) cause merosin-deficient congenital muscular dystrophy 1A (MDC1A), a complex disorder that includes hypomyelination and myodegeneration. In dystrophia muscularis (dy) mice bearing Lama2 mutations, myofibers and Schwann cells fail to assemble stable BLs, which are thought to be crucial for myofiber survival and Schwann cell differentiation. Here, we describe defects in a new allele of Lama2 in mice, nmf417, in which a point mutation substitutes Arg for Cys79 at a universally conserved CxxC motif in the laminin N-terminal (LN) domain; this domain mediates laminin-laminin interactions. nmf417 homozygosity caused progressive myodegeneration and severe peripheral amyelination in nerve roots, similar to previous Lama2 mutations, but without the pervasive BL thinning previously associated with the disorder. In direct contrast to the previously characterized dy and dy2J alleles, nmf417 homozygous myofibers frequently had thickened BLs. Severe amyelination in nmf417-mutant nerve roots suggested complete laminin 2 inactivation for Schwann cells, although myelinated fibers had normal BLs. The results reveal crucial roles for the LN domain CxxC motif in both nerve and muscle, but challenge expected relationships between LN-domain function, Ln2 activity and BL stability. The nmf417 mutation provides a defined animal model in which to investigate mechanisms and treatments for moderate forms of MDC1A.
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Referencia del producto:

Múltiplo
Nombre del producto:

Múltiplo
Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65.
Inflammation increases the abundance of inducible nitric oxide synthase (iNOS), leading to enhanced production of nitric oxide (NO), which can modify proteins by S-nitrosylation. Enhanced NO production increases the activities of the transcription factors p53 and nuclear factor κB (NF-κB) in several models of disease-associated inflammation. S-nitrosylation inhibits the activity of the protein deacetylase SIRT1. SIRT1 limits apoptosis and inflammation by deacetylating p53 and p65 (also known as RelA), a subunit of NF-κB. We showed in multiple cultured mammalian cell lines that NO donors or inflammatory stimuli induced S-nitrosylation of SIRT1 within CXXC motifs, which inhibited SIRT1 by disrupting its ability to bind zinc. Inhibition of SIRT1 reduced deacetylation and promoted activation of p53 and p65, leading to apoptosis and increased expression of proinflammatory genes. In rodent models of systemic inflammation, Parkinson's disease, or aging-related muscular atrophy, S-nitrosylation of SIRT1 correlated with increased acetylation of p53 and p65 and activation of p53 and NF-κB target genes, suggesting that S-nitrosylation of SIRT1 may represent a proinflammatory switch common to many diseases and aging.
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Referencia del producto:

Múltiplo
Nombre del producto:

Múltiplo
Expression of Na+-K+ -2Cl- cotransporter 1 is epigenetically regulated during postnatal development of hypertension.
The expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is upregulated in spontaneously hypertensive rat (SHR). We investigated whether expression of NKCC1 is epigenetically regulated during postnatal development of hypertension.The mesenteric arteries from 5-, 10-, and 18-week-old Wistar-Kyoto rats (WKY) and SHRs were subjected to vascular contraction. We determined expression levels of Nkcc1 mRNA and protein, methylation status, and histone modification of Nkcc1 promoter, and DNA methyltransferase (DNMT) activity.The inhibition of dose-response curves by bumetanide, an inhibitor of NKCC1, as well as the expression of Nkcc1 mRNA and protein was comparable between 5-week-old SHR and age-matched WKY, but greater in 18-week-old SHR than in age-matched WKY. Nkcc1 promoter in WKY was getting methylated with age whereas that in SHR mostly remained hypomethylated after development of hypertension. DNMT3B was highly associated with the promoter of WKY, whereas the CXXC finger protein 1 (Cfp1) was highly bound to the promoter of SHR. At the age of 18 weeks, the DNMT activity in aorta of WKY was about threefold higher than that of SHR. The transcription-activating histone code acetyl H3 was higher in SHR than in WKY, whereas suppressive histone code dimethyl H3K9 was greater in WKY than in SHR.It is concluded that expression of NKCC1 is epigenetically upregulated during postnatal development of hypertension. Our data indicate that maintenance of hypomethylation in Nkcc1 promoter of SHR resulting from low DNMT activity plays an important role in the upregulation of NKCC1 during development of spontaneous hypertension.
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17-371
Nombre del producto:

EZ-ChIP™
Intrinsic and extrinsic connections of Tet3 dioxygenase with CXXC zinc finger modules.
Tet proteins are emerging as major epigenetic modulators of cell fate and plasticity. However, little is known about how Tet proteins are targeted to selected genomic loci in distinct biological contexts. Previously, a CXXC-type zinc finger domain in Tet1 was shown to bind CpG-rich DNA sequences. Interestingly, in human and mouse the Tet2 and Tet3 genes are adjacent to Cxxc4 and Cxxc10-1, respectively. The CXXC domains encoded by these loci, together with those in Tet1 and Cxxc5, identify a distinct homology group within the CXXC domain family. Here we provide evidence for alternative mouse Tet3 transcripts including the Cxxc10-1 sequence (Tet3(CXXC)) and for an interaction between Tet3 and Cxxc4. In vitro Cxxc4 and the isolated CXXC domains of Tet1 and Tet3(CXXC) bind DNA substrates with similar preference towards the modification state of cytosine at a single CpG site. In vivo Tet1 and Tet3 isoforms with and without CXXC domain hydroxylate genomic 5-methylcytosine with similar activity. Relative transcript levels suggest that distinct ratios of Tet3(CXXC) isoforms and Tet3-Cxxc4 complex may be present in adult tissues. Our data suggest that variable association with CXXC modules may contribute to context specific functions of Tet proteins.
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AB9610
Nombre del producto:

Anti-Olig-2 Antibody
Recruitment of specificity protein 1 by CpG hypomethylation upregulates Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.
Promoter hypomethylation leads to upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) in the spontaneously hypertensive rat (SHR). We hypothesized that recruitment of Specificity Protein 1 (Sp1) by CpG hypomethylation would result in upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.Sham-operated Wistar-Kyoto (WKY) rats (sham) and angiotensin II (Ang II)-infused WKY rats, as well as SHRs, were used in this study. We performed real-time PCR and western blot for determination of the expression levels of Nkcc1 mRNA and protein, respectively, and bisulphite sequencing for determination of the methylation status of the proximal promoter; an assay kit was used for assessment of the activity of DNA methyltransferase (DNMT), and the electrophoretic mobility shift assay (EMSA) was used for assessment of binding of Sp1 to cis-element, and promoter function was assessed using the luciferase assay.Both Ang II-infused WKY rats and SHRs showed higher expression of Nkcc1 mRNA and protein and less DNA methylation, compared with sham. CpG methylation at Sp1 response elements interfered with binding of Sp1, resulting in disabled promoter activity. Both types of hypertensive rats showed hypomethylation of CpG dinucleotides in Sp1 response elements in accordance with the decrease of DNMT activity. DNMT3b and MeCP2 were highly recruited to the more methylated promoter of normotensive rats, whereas the CXXC finger protein 1 (Cfp1), Sp1 and RNA polymerase II were highly recruited to the less methylated promoter of hypertensive rats.Our results indicate that recruitment of Sp1 by CpG hypomethylation leads to upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.
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Referencia del producto:

17-371
Nombre del producto:

EZ-ChIP™
Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.
Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding-impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.
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Referencia del producto:

AB742
Nombre del producto:

Anti-Apolipoprotein B Antibody
Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes.
Polycomb group (PcG) proteins play important roles in repressing lineage-specific genes and maintaining the undifferentiated state of mouse embryonic stem cells (mESCs). However, how PcG proteins are recruited to their target genes is largely unknown. Here, we show that the H3K36-specific histone demethylase Kdm2b is highly expressed in mESCs and regulated by the pluripotent factors Oct4 and Sox2 directly. Depletion of Kdm2b in mESCs causes de-repression of lineage-specific genes and induces early differentiation. The function of Kdm2b depends on its CxxC-ZF domain, which mediates its genome-wide binding to CpG islands (CGIs). Kdm2b interacts with the core components of polycomb repressive complex 1 (PRC1) and recruits the complex to the CGIs of early lineage-specific genes. Thus, our study not only reveals an Oct4-Sox2-Kdm2b-PRC1-CGI regulatory axis and its function in maintaining the undifferentiated state of mESCs, but also demonstrates a critical function of Kdm2b in recruiting PRC1 to the CGIs of lineage-specific genes to repress their expression.
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Referencia del producto:

Múltiplo
Nombre del producto:

Múltiplo