Incorporation of a histone mutant with H3K56 site substitution perturbs the replication machinery in mouse embryonic stem cells.

Sense mutations in several conserved modifiable sites of histone H3 have been found to be strongly correlated with multiple tissue-specific clinical cancers. These clinical site mutants acquire a distinctively new epigenetic role and mediate cancer evolution. In this study, we mimicked histone H3 at the 56th lysine (H3K56) mutant incorporation in mouse embryonic stem cells (mESCs) by lentivirus-mediated ectopic expression and analyzed the effects on replication and epigenetic regulation. The data show that two types of H3K56 mutants, namely H3 lysine 56-to-methionine (H3K56M) and H3 lysine 56-to-alanine (H3K56A), promote replication by recruiting more minichromosome maintenance complex component 3 and checkpoint kinase 1 onto chromatin compared with wild-type histone H3 and other site substitution mutants. Under this condition, the frequency of genomic copy number gain in H3K56M and H3K56A cells globally increases, especially in the Mycl1 region, a known molecular marker frequently occurring in multiple malignant cancers. Additionally, we found the disruption of H3K56 acetylation distribution in the copy-gain regions, which indicates a probable epigenetic mechanism of H3K56M and H3K56A. We then identified that H3K56M and H3K56A can trigger a potential adaptation to transcription; genes involved in the mitogen-activated protein kinase pathway are partially upregulated, whereas genes associated with intrinsic apoptotic function show obvious downregulation. The final outcome of ectopic H3K56M and H3K56A incorporation in mESCs is an enhanced ability to form carcinomas. This work indicates that H3K56 site conservation and proper modification play important roles in harmonizing the function of the replication machinery in mESCs.

The essential roles of Mps1 in spermatogenesis and fertility in mice.

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.

Nucleotide variation in histone H2BL drives crossalk of histone modification and promotes tumour cell proliferation by upregulating c-Myc.

Gene mutations play important roles in tumour development. In this study, we identified a functional histone H2B mutation H2BL-T11C, causing an amino acid variation from Leu to Pro (L3P, H2BL-L3P). Cells overexpressing H2BL-L3P showed stronger proliferation, colony formation, tumourigenic abilities, and a different cell cycle distribution. Meanwhile, the c-Myc expression was elevated as evident by RNA-seq. We further revealed that an H2BK5ac-H2BK120ubi crosstalk which regulates gene transcription. Moreover, EdU staining demonstrated an important role of c-Myc in accelerating cell cycle progression through the G1/S checkpoint, while treatment with 10058-F4, an inhibitor of the c-Myc/MAX interaction, alleviated the abnormal cell proliferation and cell cycle distribution in vitro and partially inhibited tumour growth in vivo. The mutation of amino acid L3P is associated with tumour progression, suggesting patients carrying this SNP may have higher risk of tumour development.

Epigenetic regulation of the Warburg effect by H2B monoubiquitination.

Cancer cells reprogram their energy metabolic system from the mitochondrial oxidative phosphorylation (OXPHOS) pathway to a glucose-dependent aerobic glycolysis pathway. This metabolic reprogramming phenomenon is known as the Warburg effect, a significant hallmark of cancer. However, the detailed mechanisms underlying this event or triggering this reprogramming remain largely unclear. Here, we found that histone H2B monoubiquitination (H2Bub1) negatively regulates the Warburg effect and tumorigenesis in human lung cancer cells (H1299 and A549 cell lines) likely through controlling the expression of multiple mitochondrial respiratory genes, which are essential for OXPHOS. Moreover, our work also suggested that pyruvate kinase M2 (PKM2), the rate-limiting enzyme of glycolysis, can directly interact with H2B in vivo and in vitro and negatively regulate the level of H2Bub1. The inhibition of cell proliferation and nude mice xenograft of human lung cancer cells induced by PKM2 knockdown can be partially rescued through lowering H2Bub1 levels, which indicates that the oncogenic function of PKM2 is achieved, at least partially, through the control of H2Bub1. Furthermore, PKM2 and H2Bub1 levels are negatively correlated in cancer specimens. Therefore, these findings not only provide a novel mechanism triggering the Warburg effect that is mediated through an epigenetic pathway (H2Bub1) but also reveal a novel metabolic regulator (PKM2) for the epigenetic mark H2Bub1. Thus, the PKM2-H2Bub1 axis may become a promising cancer therapeutic target.

Histone acetyltransferase CBP-related H3K23 acetylation contributes to courtship learning in Drosophila.

BACKGROUND: Histone modifications are critical in regulating neuronal processes. However, the impacts of individual histone modifications on learning and memory are elusive. Here, we investigated the contributions of histone H3 lysine modifications to learning and memory in Drosophila by using histone lysine-to-alanine mutants. RESULTS: Behavioural analysis indicated that compared to the H3WT group, mutants overexpressing H3K23A displayed impaired courtship learning. Chromatin immunoprecipitation analysis of H3K23A mutants showed that H3K23 acetylation (H3K23ac) levels were decreased on learning-related genes. Knockdown of CREB-binding protein (CBP) decreased H3K23ac levels, attenuated the expression of learning-related genes, led to a courtship learning defect and altered development of the mushroom bodies. A decline in courtship learning ability was observed in both larvae and adult treatments with ICG-001. Furthermore, treatment of Drosophila overexpressing mutated H3K23A with a CBP inhibitor did not aggravate the learning defect. CONCLUSIONS: H3K23ac, catalysed by the acetyltransferases dCBP, contributes to Drosophila learning, likely by controlling the expression of specific genes. This is a novel epigenetic regulatory mechanism underlying neuronal behaviours.

NASP antagonize chromatin accessibility through maintaining histone H3K9me1 in hepatocellular carcinoma.

The regulation of histone deposits mediated by multi-chaperone complexes under physiological conditions remains to be further investigated. Here, we studied the function of nuclear autoantigenic sperm protein (NASP) in the regulation of liver cancer. We found that NASP levels in liver tumors were generally higher than in normal liver tissues and NASP down-regulation inhibited liver cancer cells from forming tumors. We further analyzed cellular responses and epigenetic mechanisms of the histone H3-H4 shortage induced by NASP knockdown in liver cancer cells. The results showed that the major effects of NASP knockdown were globally enhanced chromatin accessibility, which facilitates transcription release, and failure of replication initiation. Furthermore, we demonstrated that NASP depletion led to a global decrease of histone H3K9me1 modification associated with newly H3 processing, which occurred directly at the promoters of up-regulated anti-tumor genes BACH2 and RunX1T1. This also resulted in a synergistic effect on enhanced apoptosis with Myc and p53 decreases. Overall, our work provides new insights into the roles of NASP in tumorigenesis and cancer prevention.

Quantitative Dynamics of Proteome, Acetylome, and Succinylome during Stem-Cell Differentiation into Hepatocyte-like Cells.

Stem-cell differentiation is a complex biological process controlled by a series of functional protein clusters and signaling transductions, especially metabolism-related pathways. Although previous studies have quantified the proteome and phosphoproteome for stem-cell differentiation, the investigation of acylation-mediated regulation is still absent. In this study, we quantitatively profiled the proteome, acetylome, and succinylome in pluripotent human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). In total, 3843 proteins, 185 acetylation sites in 103 proteins, and 602 succinylation sites in 391 proteins were quantified. The quantitative proteome showed that in differentiated HLCs the TGF-ß, JAK-STAT, and RAS signaling pathways were activated, whereas ECM-related processes such as sulfates and leucine degradation were depressed. Interestingly, it was observed that the acetylation and succinylation were more intensive in hESCs, whereas protein processing in endoplasmic reticulum and the carbon metabolic pathways were especially highly succinylated. Because the metabolism patterns in pluripotent hESCs and the differentiated HLCs were different, we proposed that the dynamic acylations, especially succinylation, might regulate the Warburg-like effect and TCA cycle during differentiation. Taken together, we systematically profiled the protein and acylation levels of regulation in pluripotent hESCs and differentiated HLCs, and the results indicated the important roles of acylation in pluripotency maintenance and differentiation.

Histone H3K27 methylation modulates the dynamics of FANCD2 on chromatin to facilitate NHEJ and genome stability.

Dysregulation of the homeostatic balance of histone H3 di- and tri-methyl lysine 27 (H3K27me2/3) levels caused by the mis-sense mutation of histone H3 (H3K27M) is reported to be associated with various types of cancers. In this study, we found that reduction in H3K27me2/3 caused by H3.1K27M, a mutation of H3 variants found in patients with diffuse intrinsic pontine glioma (DIPG), dramatically attenuated the presence of 53BP1 (also known as TP53BP1) foci and the capability of non-homologous end joining (NHEJ) in human dermal fibroblasts. H3.1K27M mutant cells showed increased rates of genomic insertions/deletions and copy number variations, as well as an increase in p53-dependent apoptosis. We further showed that both hypo-H3K27me2/3 and H3.1K27M interacted with FANCD2, a central player in the choice of DNA repair pathway. H3.1K27M triggered the accumulation of FANCD2 on chromatin, suggesting an interaction between H3.1K27M and FANCD2. Interestingly, knockdown of FANCD2 in H3.1K27M cells recovered the number of 53BP1-positive foci, NHEJ efficiency and apoptosis rate. Although these findings in HDF cells may differ from the endogenous regulation of the H3.1K27M mutant in the specific tumor context of DIPG, our results suggest a new model by which H3K27me2/3 facilitates NHEJ and the maintenance of genome stability.This article has an associated First Person interview with the first author of the paper.

Development of Incompletely Fused Carpels in Maize Ovary Revealed by miRNA, Target Gene and Phytohormone Analysis.

Although the molecular basis of carpel fusion in maize ovary development remains largely unknown, increasing evidence suggests a critical role of microRNAs (miRNAs). In this study, a combination of miRNA sequencing, degradome and physiological analyses was used to characterize carpel fusion development in maize ovaries showing incompletely (IFC) and completely fused carpels (CFC). A total of 162 known miRNAs distributed across 33 families were identified, of which 20 were differentially expressed. In addition, 53 miRNA candidates were identified, of which 10 were differentially expressed in the IFC and CFC ovaries. In degradome analysis, a total of 113 and 11 target genes were predicted for the known and novel miRNAs, respectively. Moreover, 24 (60%) target genes of the differentially expressed known miRNAs were found to code transcription factors, including auxin response factor (ARF), TB1-CYC-PCFs (TCP), APETALA2 (AP2), growth regulating factor (GRF), MYB, NAC, and NF-YA, all of which have been shown to play a role in carpel fusion development. Correlation analysis of these differentially expressed known miRNAs and their targets with phytohormone signals revealed significant correlations with at least one phytohormone signal, the main regulator of carpel fusion development. These results suggest that incomplete carpel fusion is partly the result of differential expression of certain miRNAs and their targets. Overall, these findings improve our knowledge of the effect of miRNA regulation on target expression, providing a useful resource for further analysis of the interactions between miRNAs, target genes and phytohormones during carpel fusion development in maize.

Histone H2B monoubiquitination is a critical epigenetic switch for the regulation of autophagy.

Autophagy is an evolutionarily conserved cellular process that primarily participates in lysosome-mediated protein degradation. Although autophagy is a cytoplasmic event, how epigenetic pathways are involved in the regulation of autophagy remains incompletely understood. Here, we found that H2B monoubiquitination (H2Bub1) is down-regulated in cells under starvation conditions and that the decrease in H2Bub1 results in the activation of autophagy. We also identified that the deubiquitinase USP44 is responsible for the starvation-induced decrease in H2Bub1. Furthermore, the changes in H2Bub1 affect the transcription of genes involved in the regulation of autophagy. Therefore, this study reveals a novel epigenetic pathway for the regulation of autophagy through H2Bub1.

Differential morphology and transcriptome profile between the incompletely fused carpels ovary and its wild-type in maize.

We have isolated a new mutation in maize, incompletely fused carpels (ifc), which results in an open stylar canal on the ovary and an incomplete pericarp at the top of the kernel. The maize ovary derives from the fusion of three carpels; however, the molecular networks regulating maize carpel fusion remain largely unclear. In this study, RNA sequencing (RNA-seq) was performed on wild-type (WT) and ifc ovaries that were collected after carpel fusion defects could be morphologically distinguished. In total, 877 differentially expressed genes were identified. Functional analysis revealed overexpression of genes related to "DNA binding", "transcription regulation", "hormones", and "stress responses". Among the 88 differentially expressed transcription factor (TF) genes, five showed a high degree of conservation (77.7-88.0% amino acid identity) of their conserved domains with genes associated with carpel fusion deficiency in Arabidopsis thaliana, suggesting that these five genes might control carpel fusion in maize. In addition, 30 genes encoding components of hormone synthesis and signaling pathways were differentially expressed between ifc and WT ovaries, indicating complex hormonal regulation during carpel fusion. These results help elucidate the underlying mechanisms that regulate carpel fusion, supporting the functional analysis of genes involved in producing this phenotype.

Histone H1-mediated epigenetic regulation controls germline stem cell self-renewal by modulating H4K16 acetylation.

Epigenetics plays critical roles in controlling stem cell self-renewal and differentiation. Histone H1 is one of the most critical chromatin regulators, but its role in adult stem cell regulation remains unclear. Here we report that H1 is intrinsically required in the regulation of germline stem cells (GSCs) in the Drosophila ovary. The loss of H1 from GSCs causes their premature differentiation through activation of the key GSC differentiation factor bam. Interestingly, the acetylated H4 lysine 16 (H4K16ac) is selectively augmented in the H1-depleted GSCs. Furthermore, overexpression of mof reduces H1 association on chromatin. In contrast, the knocking down of mof significantly rescues the GSC loss phenotype. Taken together, these results suggest that H1 functions intrinsically to promote GSC self-renewal by antagonizing MOF function. Since H1 and H4K16 acetylation are highly conserved from fly to human, the findings from this study might be applicable to stem cells in other systems.

A conserved RAD6-MDM2 ubiquitin ligase machinery targets histone chaperone ASF1A in tumorigenesis.

Chromatin is a highly organized and dynamic structure in eukaryotic cells. The change of chromatin structure is essential in many cellular processes, such as gene transcription, DNA damage repair and others. Anti-silencing function 1 (ASF1) is a histone chaperone that participates in chromatin higher-order organization and is required for appropriate chromatin assembly. In this study, we identified the E2 ubiquitin-conjugating enzyme RAD6 as an evolutionary conserved interacting protein of ASF1 in D. melanogaster and H. sapiens that promotes the turnover of ASF1A by cooperating with a well-known E3 ligase, MDM2, via ubiquitin-proteasome pathway in H. sapiens. Further functional analyses indicated that the interplay between RAD6 and ASF1A associates with tumorigenesis. Together, these data suggest that the RAD6-MDM2 ubiquitin ligase machinery is critical for the degradation of chromatin-related proteins.

Microarray Analysis Reveals Potential Biological Functions of Histone H2B Monoubiquitination.

Histone H2B monoubiquitination is a key histone modification that has significant effects on chromatin higher-order structure and gene transcription. Multiple biological processes have been suggested to be tightly related to the dynamics of H2B monoubiquitination. However, a comprehensive understanding of biological roles of H2B monoubiquitination is still poorly understood. In the present study, we developed an efficient tool to disrupt endogenous H2B monoubiquitination levels by using an H2BK120R mutant construct expressed in human cells. Genome-wide microarray analysis of these cells revealed a potential global view of biological functions of H2B monoubiquitination. Bioinformatics analysis of our data demonstrated that while H2B monoubiquitination expectedly affected a number of previously reported biological pathways, we also uncovered the influence of this histone modification on many novel biological processes. Therefore, our work provided valuable information for understanding the role of H2B monoubiquitination and indicated potential directions for its further studies.

WITHDRAWN: WSTF Phosphorylation Specifically Links H3K9ac with H4K16ac through PCAF/WSTF/MOF Complex.

This article has been withdrawn by the authors.

RAD6 promotes homologous recombination repair by activating the autophagy-mediated degradation of heterochromatin protein HP1.

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.

MicroRNA-383 regulates the apoptosis of tumor cells through targeting Gadd45g.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding single-stranded RNA molecules that inhibit gene expression at post-transcriptional level. Gadd45g (growth arrest and DNA-damage-inducible 45 gamma) is a stress-response protein, which has been implicated in several biological processes, including DNA repair, the cell cycle and cell differentiation. RESULTS: In this work, we found that miR-383 is a negative regulator of Gadd45g. Forced expression of miR-383 decreased the expression of Gadd45g through binding to the 3' untranslated region (3'-UTR), whereas inhibition of miR-383 increased Gadd45g expression. The presence of miR-383 increased the cellular sensitivity to DNA damage in breast cancer cells, which was rescued by ectopic expression of Gadd45g without the 3'-UTR. miR-383 also regulates the expression of Gadd45g in embryonic stem (ES) cells, but not their apoptosis under genotoxic stress. miR-383 was further showed to negatively regulate ES cell differentiation via targeting Gadd45g, which subsequently modulates the pluripotency-associated genes. Taken together, our study demonstrates that miR-383 is a negative regulator of Gadd45g in both tumor cells and ES cells, however, has distinct function in regulating cell apoptosis. miR-383 may be used as antineoplastic agents in cancer chemotherapy. CONCLUSION: We demonstrate for the first time that miR-383 can specifically regulates the expression of Gadd45g by directly targeting to the 3-UTR region of Gadd45g mRNA, a regulatory process conserved in human tumor cells and mouse embryonic stem cells. These two compotents can be potentially used as antineoplastic agents in cancer chemotherapy.

Human RAD6 promotes G1-S transition and cell proliferation through upregulation of cyclin D1 expression.

Protein ubiquitinylation regulates protein stability and activity. RAD6, an E2 ubiquitin-conjugating enzyme, which that has been substantially biochemically characterized, functions in a number of biologically relevant pathways, including cell cycle progression. In this study, we show that RAD6 promotes the G1-S transition and cell proliferation by regulating the expression of cyclin D1 (CCND1) in human cells. Furthermore, our data indicate that RAD6 influences the transcription of CCND1 by increasing monoubiquitinylation of histone H2B and trimethylation of H3K4 in the CCND1 promoter region. Our study presents, for the first time, an evidence for the function of RAD6 in cell cycle progression and cell proliferation in human cells, raising the possibility that RAD6 could be a new target for molecular diagnosis and prognosis in cancer therapeutics.

The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method.

The cascade mechanisms of nitric oxide as a second messenger of ultraviolet B in inhibiting mesocotyl elongations.

In this report, a number of physiological aspects was examined during developmental growth of maize seedling's mesocotyl. It was found that ultraviolet B (UVB) radiation was able to significantly induce nitric oxide synthase (NOS) activities and speedup the release of apparent nitric oxide (NO) of mesocotyl and that exogenous NO donor's rhizospheric treatments may mimic the responses of the mesocotyl to UVB radiation, such as the inhibition of mesocotyl elongation, the decrease in exo- and endoglucanase activities and the increase in protein content of cell wall of mesocotyl. When the seedlings were treated with N-nitro-L-arginine, an inhibitor of NOS, the mesocotyl elongation was promoted, the exo- and endoglucanase activities were raised and the protein content was reduced. However, under UVB radiation, the effects of exogenous NO on several physiological aspects of mesocotyl were similar to those of exogenous reactive oxygen species (ROS) eliminator, N-acetyl-cysteine. All the physiological changes were associated with either the exogenous NO supply or the activities of NOS in plant. Accordingly, it is assumed that reduction in mesocotyl length caused by UVB radiation was possibly achieved through modification of the chemical properties of the cell wall polysaccharides, which was induced by NO and ROS synergically mediated changes in exo- and endo-beta-D-glucanases activities in cell walls, and NO was one of the main signaling molecule of UVB radiation in inhibiting mesocotyl elongations. So NO might function as both a second messenger and an antioxidant of UVB radiation during developmental growth of the mesocotyl.