Hbo1 is a histone acetyltransferase (HAT) that is required for global histone H4 acetylation, steroiddependent transcription, and chromatin loading of MCM2-7 during DNA replication licensing. It is the catalytic subunit of protein complexes that include ING and JADE proteins, growth regulatory factors and candidate tumor suppressors. These complexes are thought to act via tumor suppressor p53, but the molecular mechanisms and links between stress signaling and chromatin, are currently unknown. Here, we show that p53 physically interacts with Hbo1 and negatively regulates its HAT activity in vitro and in cells. Two physiological stresses that stabilize p53, hyperosmotic shock and DNA replication fork arrest, also inhibit Hbo1 HAT activity in a p53-dependent manner. Hyperosmotic stress during G 1 phase specifically inhibits the loading of the MCM2-7 complex, providing an example of the chromatin output of this pathway. These results reveal a direct regulatory connection between p53-responsive stress signaling and Hbo1-dependent chromatin pathways.The dynamic regulation of chromatin structure and function is essential for normal cell proliferation and differentiation. This regulation is mediated by several overlapping pathways, including the posttranslational enzymatic modification of histones, the alteration of nucleosome structure by DNA-dependent ATPase complexes, and changes in the histone variant composition of chromatin (4,34,58). Alterations in histone modification enzymes, particularly histone acetyltransferase (HAT) enzymes, have been linked to human cancer (23, 73). Viral oncoproteins, such as adenovirus E1A or simian virus 40 large T antigen, target a number of these enzymes, including p300, CBP, and PCAF. Furthermore, in addition to modifying histones, these HATs can also directly acetylate and activate tumor suppressors and key growth control transcription factors such as p53, Rb, and E2F (24). The MYST family of histone acetyltransferases, named for the four founding proteins in the family (67), can also contribute to carcinogenesis and tumor progression. The MYST proteins are part of large multisubunit HAT complexes conserved from yeast to humans, and they have diverse roles in gene expression, DNA replication, and DNA repair (72). The human MOZ gene, encoding one of the human MYST enzymes, was first identified as a translocation fusion with CBP in acute myeloid leukemias (9). Subsequently, a number of other translocation fusions involving the MYST HATs MOZ and MORF and partners including CBP, p300, MLL, and TIF2 have been identified. It is thought that the mislocalization or misregulation of the HAT activities of these fusions contributes to tumor formation or progression (72).Hbo1 is a member of the MYST family of HAT enzymes and is conserved from flies to humans. It has essential roles in DNA replication and transcription (1,12,22,32,55,59) and is the catalytic subunit of at least two protein complexes comprised of JADE1/JADE2/JADE3 paralogs, hEaf6, and either ING4 or ING5, two members of the "inh...

Methylation of histone tails plays a pivotal role in the regulation of a wide range of biological processes. SET and MYND domain-containing protein (SMYD) is a methyltransferase, five family members of which have been identified in humans. SMYD1, SMYD2, SMYD3, and SMYD4 have been found to play critical roles in carcinogenesis and/or the development of heart and skeletal muscle. However, the physiological functions of SMYD5 remain unknown. To investigate the function of Smyd5 in vivo, zebrafish were utilised as a model system. We first examined smyd5 expression patterns in developing zebrafish embryos. Smyd5 transcripts were abundantly expressed at early developmental stages and then gradually decreased. Smyd5 was expressed in all adult tissues examined. Loss-of-function analysis of Smyd5 was then performed in zebrafish embryos using smyd5 morpholino oligonucleotide (MO). Embryos injected with smyd5-MO showed normal gross morphological development, including of heart and skeletal muscle. However, increased expression of both primitive and definitive hematopoietic markers, including pu.1, mpx, l-plastin, and cmyb, were observed. These phenotypes of smyd5-MO zebrafish embryos were also observed when we introduced mutations in smyd5 gene with the CRISPR/Cas9 system. As the expression of myeloid markers was elevated in smyd5 loss-of-function zebrafish, we propose that Smyd5 plays critical roles in hematopoiesis.

Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.

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