Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, the presence of quiescent-like/G0 states, therefore, represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a G0-reporter to identify genes that when inhibited trap GSCs in G0-like states. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, which targets both histones (e.g., H4) and non-histone proteins for acetylation. NuA4 functions as a transcriptional activator, whose activities are coordinated with MYC in certain contexts, and also participates in DNA double-strand break repair by facilitating chromatin opening. However, currently little is known about the roles for NuA4 complex in GBM biology. Through modeling KAT5 function in GSC in vitro cultures and in vivo tumors, we find that KAT5 inhibition causes cells to arrest in a G0-like state with high p27 levels, G1-phase DNA content, low protein synthesis rates, low rRNA rates, lower metabolic rate, suppression of cell cycle gene expression, and low histone H4 acetylation. Interestingly, partial inhibition of KAT5 activity slows highly aggressive tumor growth, while increasing p27hi H4-aclow populations. Remarkably, we that low grade gliomas have significantly higher H4-aclow subpopulations and generally lower H4-ac levels than aggressive grade IV tumors. Taken together, our results suggest that NuA4/KAT5 activity may play a key role in quiescence ingress/egress in glioma and that targeting its activity in high grade tumors may effectively “down grade” them, thus, increase patient survival.

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Protein kinase D1 phosphorylation of KAT7 enhances its protein stability and promotes replication licensing and cell proliferation

Cell Death Discovery ◽

10.1038/s41420-020-00323-w ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Yao Liang ◽

Yuanyuan Su ◽

Chenzhong Xu ◽

Na Zhang ◽

Doudou Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Replication ◽

Protein Kinase ◽

Histone H4 ◽

Replication Complex ◽

Protein Kinase D1 ◽

Defective Mutant ◽

Histone H4 Acetylation

Abstract The histone acetyltransferase (HAT) KAT7/HBO1/MYST2 plays a crucial role in the pre-replication complex (pre-RC) formation, DNA replication and cell proliferation via acetylation of histone H4 and H3. In a search for protein kinase D1 (PKD1)-interacting proteins, we have identified KAT7 as a potential PKD1 substrate. We show that PKD1 directly interacts and phosphorylates KAT7 at Thr97 and Thr331 in vitro and in vivo. PKD1-mediated phosphorylation of KAT7 enhances its expression levels and stability by reducing its ubiquitination-mediated degradation. Significantly, the phospho-defective mutant KAT7-Thr97/331A attenuates histone H4 acetylation levels, MCM2/6 loading on the chromatin, DNA replication and cell proliferation. Similarly, PKD1 knockdown decreases, whereas the constitutive active mutant PKD1-CA increases histone H4 acetylation levels and MCM2/6 loading on the chromatin. Overall, these results suggest that PKD1-mediated phosphorylation of KAT7 may be required for pre-RC formation and DNA replication.

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MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽

10.1530/rep-19-0334 ◽

2020 ◽

Vol 159 (1) ◽

pp. 1-13 ◽

Cited By ~ 4

Author(s):

Wei Cui ◽

Agnes Cheong ◽

Yongsheng Wang ◽

Yuran Tsuchida ◽

Yong Liu ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Cell Number ◽

Blastocyst Stage ◽

Histone H4 ◽

Histone H4 Acetylation ◽

Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

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TFIIH-Associated Cdk7 Kinase Functions in Phosphorylation of C-Terminal Domain Ser7 Residues, Promoter-Proximal Pausing, and Termination by RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.00637-09 ◽

2009 ◽

Vol 29 (20) ◽

pp. 5455-5464 ◽

Cited By ~ 184

Author(s):

Kira Glover-Cutter ◽

Stéphane Larochelle ◽

Benjamin Erickson ◽

Chao Zhang ◽

Kevan Shokat ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Histone H4 ◽

Pol Ii ◽

Terminal Domain ◽

Histone H4 Acetylation ◽

Flanking Regions

ABSTRACT The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7 as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5′ ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase “leaking” into the gene. Consistent with a 5′ pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36—two marks of elongation—within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3′ ends, it was detected within genes and 3′-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

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Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay

Biochemical Journal ◽

10.1042/bj20080536 ◽

2008 ◽

Vol 413 (1) ◽

pp. 143-150 ◽

Cited By ~ 12

Author(s):

Dennis Wegener ◽

Christian Hildmann ◽

Daniel Riester ◽

Andreas Schober ◽

Franz-Josef Meyer-Almes ◽

...

Keyword(s):

Gene Expression ◽

Histone Deacetylases ◽

Histone Deacetylase Inhibitors ◽

Cell Metabolism ◽

Hdac Inhibitors ◽

Tumour Suppressor Genes ◽

Histone H4 ◽

Histone H4 Acetylation

HDACs (histone deacetylases) are considered to be among the most important enzymes that regulate gene expression in eukaryotic cells. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels are linked to repression of gene expression. HDACs associate with a number of cellular oncogenes and tumour-suppressor genes, leading to an aberrant recruitment of HDAC activity, which results in changes of gene expression, impaired differentiation and excessive proliferation of tumour cells. Therefore HDAC inhibitors are efficient anti-proliferative agents in both in vitro and in vivo pre-clinical models of cancer, making them promising anticancer therapeutics. In the present paper, we present the results of a medium-throughput screening programme aiming at the identification of novel HDAC inhibitors using HDAH (HDAC-like amidohydrolase) from Bordetella or Alcaligenes strain FB188 as a model enzyme. Within a library of 3719 compounds, several new classes of HDAC inhibitor were identified. Among these hit compounds, there were also potent inhibitors of eukaryotic HDACs, as demonstrated by an increase in histone H4 acetylation, accompanied by a decrease in tumour cell metabolism in both SHEP neuroblastoma and T24 bladder carcinoma cells. In conclusion, screening of a compound library using FB188 HDAH as model enzyme identified several promising new lead structures for further development.

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210 POST-TRANSLATIONAL MODIFICATION OF HISTONE H4 ACETYLATION DURING IN VITRO MATURATION OF CANINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab210 ◽

2016 ◽

Vol 28 (2) ◽

pp. 236 ◽

Cited By ~ 1

Author(s):

T. F. Motheo ◽

D. R. Arnold ◽

W. R. R. Vicente ◽

B. I. Macente ◽

F. G. F. Filgueira ◽

...

Keyword(s):

In Vitro Maturation ◽

Least Square ◽

Penicillin G ◽

In Vitro Fertilisation ◽

Histone H4 ◽

Post Translational Modification ◽

Germinal Vesicle Breakdown ◽

Meiotic Process ◽

Histone H4 Acetylation

Proper in vitro oocyte maturation, crucial for in vitro fertilisation, is poorly understood and inefficient in dogs. Post-translational modifications of histones are involved in the meiotic process and preparation of oocytes for fertilisation. The present study aimed to evaluate post-translational histone H4 acetylation at lysine 12 (H4K12ac) and 16 (H4K16ac) in canine oocytes during in vitro maturation (IVM). Ovaries were retrieved from 1–7 year-old bitches undergoing routine ovariohysterectomy (OH). Grade I cumulus-oocyte complexes (COC) were recovered and evaluated before and after IVM in TCM 199 + 0.025 mM sodium pyruvate + 100 000 IU mL–1 penicillin G sodium + 100 mg mL–1 streptomycin sulfate, 0.003 g mL–1 BSA + 50 μL mL–1 Pluset® (500 UI FSH + 500 UI LH) + 1 μL mL–1 insulin-like growth factor (IGF) at 38.5°C in 5% CO2 for 72 h. After IVM, the nuclear stage of oocytes was established using Hoechst 33342 staining, and the acetylation patterns were investigated by indirect immunofluorescence staining of fixed immature and post-IVM oocytes (4% paraformaldehyde) with specific antibodies against the acetylated lysine residues, H4K12ac and H4K16ac. All stages were collected and evaluated simultaneously, and the experiment was repeated 4 times, with a total of 5–14 of oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIH ImageJ software 1.4, and data are presented as a percentage of the average fluorescence intensity of each specific antibody over the intensity of DNA, as determined by Hoescht staining. Immunofluorescence values were analysed using the least square ANOVA by the general linear model procedures of SAS (SAS Institute, Cary, NC, USA), and comparisons of means were further performed by Fisher’s Exact test. A probability level of P < 0.05 was considered significant. Our results indicate a variation in acetylation levels of H4K12 (P < 0.02) and H4K16 (P < 0.04) during meiosis. Fluorescence levels of H4K12ac decreased significantly when comparing immature (2.19 ± 0.62) with oocytes in metaphase I/II (MI/MII) (0.52 ± 0.14). Similarly, H4K16ac displayed a greater acetylation pattern in immature oocytes (1.20 ± 0.27) when compared to oocytes in germinal vesicle breakdown (GVBD; 0.58 ± 0.16) and MI/MII (0.62 ± 0.07) stages. These results support the hypothesis that, similar to other domestic species, post-translational modification of histone acetylation takes place during the meiotic process of oocyte IVM. Whether these histone modifications take place in a similar fashion during IVM remains to be investigated.

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Nickel Ions Increase Histone H3 Lysine 9 Dimethylation and Induce Transgene Silencing

Molecular and Cellular Biology ◽

10.1128/mcb.26.10.3728-3737.2006 ◽

2006 ◽

Vol 26 (10) ◽

pp. 3728-3737 ◽

Cited By ~ 141

Author(s):

Haobin Chen ◽

Qingdong Ke ◽

Thomas Kluz ◽

Yan Yan ◽

Max Costa

Keyword(s):

Chinese Hamster ◽

Nuclear Extract ◽

Transgene Silencing ◽

Histone H4 ◽

Nickel Ion ◽

Nickel Ions ◽

Histone H4 Acetylation ◽

H3k9 Dimethylation

ABSTRACT We have previously reported that carcinogenic nickel compounds decreased global histone H4 acetylation and silenced the gpt transgene in G12 Chinese hamster cells. However, the nature of this silencing is still not clear. Here, we report that nickel ion exposure increases global H3K9 mono- and dimethylation, both of which are critical marks for DNA methylation and long-term gene silencing. In contrast to the up-regulation of global H3K9 dimethylation, nickel ions decreased the expression and activity of histone H3K9 specific methyltransferase G9a. Further investigation demonstrated that nickel ions interfered with the removal of histone methylation in vivo and directly decreased the activity of a Fe(II)-2-oxoglutarate-dependent histone H3K9 demethylase in nuclear extract in vitro. These results are the first to show a histone H3K9 demethylase activity dependent on both iron and 2-oxoglutarate. Exposure to nickel ions also increased H3K9 dimethylation at the gpt locus in G12 cells and repressed the expression of the gpt transgene. An extended nickel ion exposure led to increased frequency of the gpt transgene silencing, which was readily reversed by treatment with DNA-demethylating agent 5-aza-2′-deoxycytidine. Collectively, our data strongly indicate that nickel ions induce transgene silencing by increasing histone H3K9 dimethylation, and this effect is mediated by the inhibition of H3K9 demethylation.

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Activation Domain-Specific and General Transcription Stimulation by Native Histone Acetyltransferase Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.855 ◽

1999 ◽

Vol 19 (1) ◽

pp. 855-863 ◽

Cited By ~ 110

Author(s):

Keiko Ikeda ◽

David J. Steger ◽

Anton Eberharter ◽

Jerry L. Workman

Keyword(s):

Histone Acetyltransferase ◽

Regulation Of Transcription ◽

Dependent Manner ◽

Saga Complex ◽

Histone H4 ◽

Activation Domain ◽

Activation Domains ◽

Nucleosomal Array ◽

Histone H4 Acetylation

ABSTRACT Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

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UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1048-1061.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1048-1061

Author(s):

I J Lee ◽

L Tung ◽

D A Bumcrot ◽

E S Weinberg

Keyword(s):

Binding Site ◽

Sea Urchin ◽

Transcriptional Start Site ◽

Sodium Dodecyl ◽

Nuclear Extract ◽

Dnase I ◽

Histone H4 ◽

Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

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Epigenetic control via allosteric regulation of mammalian protein arginine methyltransferases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706978114 ◽

2017 ◽

Vol 114 (38) ◽

pp. 10101-10106 ◽

Cited By ~ 21

Author(s):

Kanishk Jain ◽

Cyrus Y. Jin ◽

Steven G. Clarke

Keyword(s):

Cancer Metastasis ◽

Gene Activation ◽

Kinetic Studies ◽

Arginine Methylation ◽

Histone H4 ◽

Protein Arginine Methyltransferases ◽

Wide Range ◽

Substrate Concentrations

Arginine methylation on histones is a central player in epigenetics and in gene activation and repression. Protein arginine methyltransferase (PRMT) activity has been implicated in stem cell pluripotency, cancer metastasis, and tumorigenesis. The expression of one of the nine mammalian PRMTs, PRMT5, affects the levels of symmetric dimethylarginine (SDMA) at Arg-3 on histone H4, leading to the repression of genes which are related to disease progression in lymphoma and leukemia. Another PRMT, PRMT7, also affects SDMA levels at the same site despite its unique monomethylating activity and the lack of any evidence for PRMT7-catalyzed histone H4 Arg-3 methylation. We present evidence that PRMT7-mediated monomethylation of histone H4 Arg-17 regulates PRMT5 activity at Arg-3 in the same protein. We analyzed the kinetics of PRMT5 over a wide range of substrate concentrations. Significantly, we discovered that PRMT5 displays positive cooperativity in vitro, suggesting that this enzyme may be allosterically regulated in vivo as well. Most interestingly, monomethylation at Arg-17 in histone H4 not only raised the general activity of PRMT5 with this substrate, but also ameliorated the low activity of PRMT5 at low substrate concentrations. These kinetic studies suggest a biochemical explanation for the interplay between PRMT5- and PRMT7-mediated methylation of the same substrate at different residues and also suggest a general model for regulation of PRMTs. Elucidating the exact relationship between these two enzymes when they methylate two distinct sites of the same substrate may aid in developing therapeutics aimed at reducing PRMT5/7 activity in cancer and other diseases.

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