Relationship: 1709: Histone deacetylase inhibition leads to Histone acetylation, increase

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The relationship between HDAC inhibition and increase in histone acetylation is conceivably well conserved among various species including mammals.

• Hyperacetylation by HDIs such as SAHA and Cpd-60 are observed in mice (Mus musculus) [Schroeder et al., 2013].
• TSA induces acetylation of histone H4 in a time-dependent manner in mouse cell lines (Mus musculus) [Alberts et al., 1998].
• AR-42, a novel HDI, induces hyperacetylation in human pancreatic cancer cells (Homo sapiens) [Henderson et al., 2016].
• SAHA and MS-275 lead to the hyperacetylation of lysine residues of histones in human cell lines of epithelial (A549) and lymphoid origin (Jurkat) (Homo sapiens) [Choudhary et al., 2009].
• SAHA treatment induces the H3 and H4 histone acetylation in human corneal fibroblasts and conjunctiva from rabbits after glaucoma filtration surgery (Homo sapiens, Oryctolagus cuniculus) [Sharma et al., 2016].
• TSA induces the acetylation of histones H3 and H4 in Brassica napus microspore cultures (Brassica napu) [Li et al., 2014].

Key Event Relationship Description

The HDAC inhibitors (HDIs) inhibit deacetylation of the histone, leading to the increase in histone acetylation and gene transcription. HDACs deacetylate acetylated histone in epigenetic regulation [Falkenberg and Johnstone, 2014].

Histone acetylation is one of the major epigenetic mechanisms and belongs to the posttranslational modifications of histones. Histone acetyltransferase is setting the mark, and deacetylase (HDAC) is responsible for removing the acetyl group from specific lysine residues of the histones. It has been shown that the inhibition of HDACs leads to a hyperacetylation of histones and in general to an imbalance of other histone modifications.

Evidence Supporting this KER

HDACs are important proteins in the epigenetic regulation of gene transcription. Upon the inhibition of HDAC by HDIs, lysine in histone remains acetylated which leads to transcriptional activation or repression, changes in DNA replication, and DNA damage repair [Wade et al., 2008].

In all eukaryotes, the DNA containing the genetic information of an organism is organized in chromatin. The basic unit of chromatin is the nucleosome around which the naked DNA is wrapped. A nucleosome consists of two copies of each of the core histones H2A, H2B, H3, and H4 [Luger et al., 1997]. In order to dynamically regulate this highly complex structure several mechanisms are involved, including the posttranslational modifications of histones (reviewed in [Bannister and Kouzarides, 2011; Kouzarides, 2007]. For a long time, it is known that histones get acetylated and that this reaction is catalyzed by histone acetyltransferases (HAT) whereas the acetyl groups are removed by histone deacetylases (HDAC). Inhibition of HDACs by small-molecule compounds leads to hyperacetylation of the histones as the homeostasis of acetylation and deacetylation is disturbed (reviewed in [Gallinari et al., 2007]).

The major empirical evidence came from the incubation of cell culture cells with small molecule compounds that inhibit HDAC enzymes followed by western blots or acid urea gel analysis. The first evidence was shown by Riggs et al. who showed that incubation of HeLa cells with n-butyrate leads to an accumulation of acetylated histone proteins [Riggs et al., 1977]. Later, it was shown that n-butyrate specifically increases the acetylation of histone by the incorporation of radioactive [³H]acetate and analysis of the histones on acid urea gels that allow the detection of acetylated histones [Cousens et al., 1979]. TSA was shown to be an HDAC inhibitor by acid urea gel analysis in 1990 [Yoshida et al., 1990] and good evidence for VPA as an HDAC inhibitor in vitro and in vivo was shown using acetyl-specific antibodies and western blot [Gottlicher et al., 2001].

There exist several pieces of evidence showing the link between histone deacetylase inhibition and increase in histone acetylation as follows:

• Exposure of mouse embryos in utero to VPA and TSA (two well-known HDAC inhibitors) showed an increased histone acetylation level in whole embryo extracts and was also shown in situ immuno-stainings [Menegola et al., 2005].
• HDAC inhibition by HDIs leads to hyperacetylation of histone and a large number of cellular proteins such as NF-kappaB [Falkenberg and Johnstone, 2014; Chen et al., 2018].
• The concentrations of half-maximum inhibitory effect (IC₅₀) for HDAC enzyme inhibition of 20 valproic acid derivatives correlated with teratogenic potential of the compounds, and HDAC inhibitory compounds showed hyperacetylation of core histone 4 in treated F9 cells [Eikel et al., 2006].
• HDIs increase histone acetylation in the brain [Schroeder et al., 2013].
• More acetylation sites on the histones H3 and H4 are responsive to SAHA than to MS-275 indicating that an HDI selectivity exists [Choudhary et al., 2009].
• HDI AR-42 induces acetylation of histone H3 in a dose-response manner in human pancreatic cancer cell lines [Henderson et al., 2016].
• MAA treatment in rats (650 mg/kg, for 4, 8, 12, and 24 hrs) induced hyperacetylation in histones H3 and H4 of testis nuclei [Wade et al., 2008].
• HDAC inhibition induced by valproic acid (VPA) leads to histone hyperacetylation in mouse teratocarcinoma cell line F9 [Eikel et al., 2006].
• Hyperacetylation of histone H3 was observed in HDAC1-deficient ES cells [Lagger et al., 2002].
• The treatment of MAA induced histone acetylation in H3K9Ac and H4K12Ac, as well as p53K379Ac [Dayan and Hales, 2014].

HDACs affect a large number of cellular proteins including histones, which reminds us the HDAC inhibition by HDIs hyperacetylates cellular proteins other than histones and exhibit additional biological effects. It is also noted that HDAC functions as the catalytic subunits of the large protein complex, which suggests that the inhibition of HDAC by HDIs affects the function of the large multiprotein complexes of HDAC [Falkenberg and Johnstone, 2014].  Related-analysis are usually indirect or in purified systems, therefore a direct cause-consequence relation is difficult to obtain.

Quantitative Understanding of the Linkage

To quantify acetylation by HDAC, stable isotope labeling with amino acids in cell culture (SILAC) method is used [Choudhary et al., 2009].

SAHA or MS-275 treatment leads to an increase in acetylation of specific lysine residues on histones more than two-fold [Choudhary et al., 2009]. Acetylation of the variant histone H2AZ-a mark for DNA damage sites- was upregulated almost 20-fold by SAHA, whereas a number of sites on the core histones H3 and H4 are several times more highly regulated in response to SAHA than by MS-275 [Choudhary et al., 2009].

TSA (100 ng/ml) treatment leads to accumulation of the acetylated histones in a variety of mammalian cell lines, and the partially purified HDAC from wild-type FM3A cells was effectively inhibited by TSA (K_i = 3.4 nM) [Yoshida et al., 1990].

References

Alberts, A.S. et al. (1998), "Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation", Cell 92:475-487

Bannister, A. J. and Kouzarides, T. (2011), "Regulation of chromatin by histone modifications", Cell Res 21:381-395

Chen, S. et al. (2018), "Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-kB pathway dependent of HDAC3", J Neuroinflammation 15:150

Choudhary, C. et al. (2009), "Lysine acetylation targets protein complexes and co-regulates major cellular functions", Science 325:834-840

Cousens, L. S. et al. (1979), "Different accessibilities in chromatin to histone acetylase", J Biol Chem 254:1716-1723

Dayan, C. and Hales, B.F. (2014), "Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro", Birth Defects Res (Part B) 101:254-261

Eikel, D. et al. (2006), "Teratogenic effects mediated by inhibition of histone deacetylases: evidence from quantitative structure activity relationships of 20 valproic acid derivatives", Chem Res Toxicol 19:272-278

Falkenberg, K.J. and Johnstone, R.W. (2014), "Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders", Nat Rev Drug Discov 13:673-691

Gallinari, P. et al. (2007), "HDACs, histone deacetylation and gene transcription: From molecular biology to cancer therapeutics", Cell Res 17:195-211

Gottlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969-6978

Henderson, S.E. et al. (2016), "Suppression of tumor growth and muscle wasting in a transgenic mouse model of pancreatic cancer by the novel histone deacetylase inhibitor AR-42", Neoplasia 18:765-774

Kouzarides, T. (2007), "Chromatin modifications and their function", Cell 128:693-705

Lagger, G. et al. (2002), "Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression", EMBO J 21:2672-2681

Li, H. et al. (2014), "The histone deacetylase inhibitor trichostatin A promotes totipotentcy in the male gametophyte", Plant Cell 26:195-209

Luger, K. et al. (1997), "Crystal structure of the nucleosome core particle at 2.8 a resolution", Nature 389:251-260

Menegola, E. et al. (2005), "Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity", Birth Defects Res B Dev Reprod Toxicol 74:392-398

Riggs, M.G. et al. (1977), "N-butyrate causes histone modification in HeLa and friend erythroleukaemia cells", Nature 268:462-464

Schroeder, F.A. et al. (2013), "A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests", PLoS One 8:e71323

Sharma, A. et al. (2016), "Epigenetic modification prevents excessive wound healing and scar formation after glaucoma filtration surgery", Invest Ophthalmol Vis Sci 57:3381-3389

Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831

Yoshida, M. et al. (1990), "Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A", J Biol Chem 265:17174-17179

Relationship: 1806: Histone acetylation, increase leads to Altered, Gene Expression

AOPs Referencing Relationship

Key Event Relationship Description

The structure of chromatin is a major component of gene regulation in eukaryotes by providing or preventing accessibility for the transcriptional machinery to the relevant regulatory DNA sequences. Histone acetylation is one of the major posttranslational modifications that are involved in the regulation of gene expression. Generally spoken, acetylation is correlated with actively transcribed genes, whereas hypoacetylation is involved in gene silencing.

Evidence Supporting this KER

In all eukaryotes, the DNA containing the genetic information of an organism is organized in chromatin. The basic unit of chromatin is the nucleosome around which the naked DNA is wrapped. A nucleosome consists of two copies of each of the core histones H2A, H2B, H3 and H4 (Luger et al., 1997). In general, chromatin is a permissive structure for all DNA-dependent processes such as DNA replication, recombination, repair, and transcription and therefore also for gene expression. However, chromatin structure is very dynamically regulated and can be made accessible for the transcriptional machinery and is, therefore, an important mechanism of gene regulation. One mechanism of chromatin structural regulation is the post-translational modifications of the histone proteins including the acetylation of lysine residues (reviewed in (Bannister and Kouzarides, 2011; Bannister et al., 2002; Kouzarides, 2007; Tessarz and Kouzarides, 2014)). These modifications serve as a docking station for further proteins and protein complexes that finally open or close the chromatin structure and allow or inhibit access of the transcriptional machinery (Musselman et al., 2012) or directly influence DNA histone interaction (reviewed in (Tessarz and Kouzarides, 2014). Histones get acetylated by histone acetyltransferases (HAT) and deacetylated by histone deacetylases (HDAC) (reviewed in (Gallinari et al., 2007; Bannister and Kouzarides, 2011; Kouzarides, 2007)). In general, it can be assumed, hyperacetylated histones are associated with actively transcribed genes, whereas hypoacetylation of histones is involved in gene silencing.

The first direct evidence that histone acetylation has an impact on gene expression came from mutation studies in yeast. Using ChIP on chip analysis showed that mutation or deletions of HDAC enzymes lead to changed gene expression levels on a subset of genes (Xu et al., 2005; Bernstein et al., 2000; Robyr et al., 2002).

In the Drosophila cell line S2, it was shown that deregulation of transcription occurs only by know-down (RNAi) HDAC enzymes, that at least class 1 and 3 HDAC enzymes have an influence on gene expression (measured via gene chips). However, this study did not show a direct link between histone acetylation and gene expression (Foglietti et al., 2006).

In mice knockout of HDACs are mostly embryonically lethal. However, the use of embryonic stem cells and the expression and acetylation profiles shows that also in mice an imbalance of histone acetylation may lead to changes in gene expression (Zupkovitz et al., 2006).

Major gene expression changes were observed during the differentiation of hESC towards neuroectodermal progenitor cells. In these studies also the acetylation status of the deregulated genes was investigated by chromatin immunoprecipitation (Balmer et al., 2014; Balmer et al., 2012)

All above-mentioned analysis are indirect or in purified systems.
Therefore a direct cause-consequence relation is difficult to obtain.

References

Balmer, N. V., Weng, M. K., Zimmer, B. et al. (2012). Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome. Hum Mol Genet 21, 4104-4114. doi:10.1093/hmg/dds239

Balmer, N. V., Klima, S., Rempel, E. et al. (2014). From transient transcriptome responses to disturbed neurodevelopment: Role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects. Arch Toxicol 88, 1451-1468. doi:10.1007/s00204-014-1279-6

Bannister, A. J., Schneider, R. and Kouzarides, T. (2002). Histone methylation: Dynamic or static? Cell 109, 801-806. doi:S0092867402007985 [pii]

Bannister, A. J. and Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Res 21, 381-395. doi:10.1038/cr.2011.22

Bernstein, B. E., Tong, J. K. and Schreiber, S. L. (2000). Genomewide studies of histone deacetylase function in yeast. Proc Natl Acad Sci U S A 97, 13708-13713. doi:10.1073/pnas.250477697

Foglietti, C., Filocamo, G., Cundari, E. et al. (2006). Dissecting the biological functions of drosophila histone deacetylases by rna interference and transcriptional profiling. J Biol Chem 281, 17968-17976. doi:10.1074/jbc.M511945200

Gallinari, P., Di Marco, S., Jones, P. et al. (2007). Hdacs, histone deacetylation and gene transcription: From molecular biology to cancer therapeutics. Cell Res 17, 195-211. doi:7310149 [pii]

10.1038/sj.cr.7310149

Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705.

Musselman, C. A., Lalonde, M. E., Cote, J. et al. (2012). Perceiving the epigenetic landscape through histone readers. Nat Struct Mol Biol 19, 1218-1227. doi:10.1038/nsmb.2436

Robyr, D., Suka, Y., Xenarios, I. et al. (2002). Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases. Cell 109, 437-446.

Tessarz, P. and Kouzarides, T. (2014). Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol 15, 703-708. doi:10.1038/nrm3890

Xu, F., Zhang, K. and Grunstein, M. (2005). Acetylation in histone h3 globular domain regulates gene expression in yeast. Cell 121, 375-385. doi:10.1016/j.cell.2005.03.011

Zupkovitz, G., Tischler, J., Posch, M. et al. (2006). Negative and positive regulation of gene expression by mouse histone deacetylase 1. Mol Cell Biol 26, 7913-7928. doi:10.1128/MCB.01220-06

Relationship: 1807: Altered, Gene Expression leads to Altered differentiation

AOPs Referencing Relationship

Key Event Relationship Description

During early embryonic development of the nervous system-specific developmental genes need to be activated in a highly regulated and concerted manner. Genes need to be expressed (or silenced) at the right time and space. If this concerted regulation is disturbed this may lead to severe malformations in the later fetus. The type of malformation depends strongly on the timing of disturbance, i.e. during which developmental stage the disturbance happened. For the development of neural tube defects, especially neural tube closure, the disturbance must happen before neurulation at the stage when neuroepithelial cells arise. Therefore, for this AOP the imbalance of gene expression must occur before neural tube closure. However, compare AOP23 the inhibition of neural crest migration by HDAC inhibition happens later in embryonic development.

Evidence Supporting this KER

Gene expression during embryonic development is a highly regulated process. The genes need to be expressed and silenced at the right time and place. Therefore, if cells that are determined to differentiate into e.g. NEP wrongly express neural crest marker genes this may lead to a failure of neural tube closure. One reason for this effect is that these cells lose their epithelial character and undergo an epithelial mesenchymal transition as neural crest cells do (Park and Gumbiner, 2010). In summary, imbalanced gene expression may lead to the differentiation of the wrong cell type at the wrong time and space.

It was shown that HDAC inhibition changes the gene expression pattern during the differentiation of hESC towards NEP cells. Furthermore, the changed differentiation track points to a wrong differentiation towards neural crest cells (Balmer et al., 2014).

The differentiation track of hESC towards NEP is strongly disturbed in vitro by 6 different (structurally not related) HDAC inhibitors in a comparable manner (Rempel et al., 2015).

TSA treated chicken embryos showed a disturbed gene expression pattern of the posterior neural tube, that points to a wrong differentiation track towards neural crest cells (Murko et al., 2013).

References

Balmer, N. V., Klima, S., Rempel, E. et al. (2014). From transient transcriptome responses to disturbed neurodevelopment: Role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects. Arch Toxicol 88, 1451-1468. doi:10.1007/s00204-014-1279-6

Murko, C., Lagger, S., Steiner, M. et al. (2013). Histone deacetylase inhibitor trichostatin a induces neural tube defects and promotes neural crest specification in the chicken neural tube. Differentiation 85, 55-66. doi:10.1016/j.diff.2012.12.001

Park, K. S. and Gumbiner, B. M. (2010). Cadherin 6b induces bmp signaling and de-epithelialization during the epithelial mesenchymal transition of the neural crest. Development 137, 2691-2701. doi:10.1242/dev.050096

Rempel, E., Hoelting, L., Waldmann, T. et al. (2015). A transcriptome-based classifier to identify developmental toxicants by stem cell testing: Design, validation and optimization for histone deacetylase inhibitors. Arch Toxicol 89, 1599-1618. doi:10.1007/s00204-015-1573-y

Relationship: 1808: Altered differentiation leads to Neural tube defects

AOPs Referencing Relationship

Key Event Relationship Description

During the process of neural tube closure, the cells at the dorsal boundary a new cell type arises, the neural crest cells. These cells undergo an epithelial to mesenchymal transition that enables them to migrate away from the neural tube. If the neuroepithelial cells of the neural tube express the wrong regulator genes it may happen that they acquire such neural crest characteristics, which prevents them from performing the closure of the neural tube.

Evidence Supporting this KER

Many HDAC inhibitors used as drugs have been shown to induce congenital malformations, including neural tube closure defects in humans and model organisms (Menegola et al., 1996; Nau, 1994). Most studies show that after treatment with HDAC inhibitors these malformations occur in the experimental animals, however direct evidence that neural tube closure defects result from wrongly differentiated neural tube cells.

TSA treated chicken embryos showed a disturbed gene expression pattern of the posterior neural tube, that points to a wrong differentiation track towards neural crest cells. This was shown by in situ immune staining using neural crest-specific antibodies (Murko et al., 2013).

Further Menegola et al. showed direct evidence that HDAC inhibition is occurring in vivo in the neural tube of mice (Menegola et al., 2005).

Due to lacking studies directly showing that the neural tube cells are wrongly differentiated and that this may causative for closure defects the uncertainty of this KER is very high and based on correlation studies.

References

Menegola, E., Broccia, M. L., Nau, H. et al. (1996). Teratogenic effects of sodium valproate in mice and rats at midgestation and at term. Teratog Carcinog Mutagen 16, 97-108.​ doi:10.1002/(SICI)1520-6866(1996)16:2<97::AID-TCM4>3.0.CO;2-A

Menegola, E., Di Renzo, F., Broccia, M. L. et al. (2005). Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity. Birth Defects Res B Dev Reprod Toxicol 74, 392-398. doi:10.1002/bdrb.20053

Murko, C., Lagger, S., Steiner, M. et al. (2013). Histone deacetylase inhibitor trichostatin a induces neural tube defects and promotes neural crest specification in the chicken neural tube. Differentiation 85, 55-66. doi:10.1016/j.diff.2012.12.001

Nau, H. (1994). Valproic acid-induced neural tube defects. Ciba Found Symp 181, 144-152; discussion 152-160.