Tos4 mediates gene expression homeostasis through interaction with HDAC complexes independently of H3K56 acetylation.

Saccharomyces cerevisiae exhibits gene expression homeostasis, which is defined as the buffering of transcription levels against changes in DNA copy number during the S phase of the cell cycle. It has been suggested that S. cerevisiae employs an active mechanism to maintain gene expression homeostasis through Rtt109-Asf1-dependent acetylation of histone H3 on lysine 56 (H3K56). Here, we show that gene expression homeostasis can be achieved independently of H3K56 acetylation by Tos4 (Target of Swi6-4). Using Nanostring technology, we establish that Tos4-dependent gene expression homeostasis depends on its forkhead-associated (FHA) domain, which is a phosphopeptide recognition domain required to bind histone deacetylases (HDACs). We demonstrate that the mechanism of Tos4-dependent gene expression homeostasis requires its interaction with the Rpd3L HDAC complex. However, this is independent of Rpd3's well-established roles in both histone deacetylation and controlling the DNA replication timing program, as established by deep sequencing of Fluorescence-Activated Cell Sorted (FACS) S and G2 phase populations. Overall, our data reveals that Tos4 mediates gene expression homeostasis through its FHA domain-dependent interaction with the Rpd3L complex, which is independent of H3K56ac.

Detection of CALR Mutation in Clonal and Nonclonal Hematologic Diseases Using Fragment Analysis and Next-Generation Sequencing.

OBJECTIVES: To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene. METHODS: Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases. CALR screening was primarily performed using fragment analysis polymerase chain reaction, then next-generation sequencing and Sanger sequencing. RESULTS: Among the 18 patients diagnosed with clonal diseases, one had acute myeloid leukemia (positive for trisomy 8), and 17 had myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV). Patients with CML were positive for the BCR-ABL1 fusion. Ten patients were positive for JAK2 (PMF, n = 1; ET, n = 2; PV, n = 7), and three were CALR positive (ET, n = 1; PMF, n = 2). Patients diagnosed with a nonclonal disease were negative for JAK2, BCR-ABL, and CALR mutations. CONCLUSIONS: Screening for CALR mutations is essential in BCR-ABL-negative MPNs since it not only provides valuable diagnostic and prognostic information but also identifies potential treatment targets. Since this study describes the importance of screening for known and novel biomarkers, we described in detail three methods that could be easily integrated into a clinical laboratory.