InteractiveFly: GeneBrief

Histone deacetylase 3: Biological Overview | References

BIOLOGICAL OVERVIEW

Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. This study has demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster results in a reduction in body size. Further genetic studies showed that Hdac3 counteracts the overgrowth induced by InR, PI3K or S6K over-expression, and the growth regulation by Hdac3 is mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the over-expression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, results in changes in body size. Furthermore, H4K16ac was found to be modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway results in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracts the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, these studies indicated that Hdac3 serves as an important regulator of the PI3K pathway and reveals a novel link between histone acetylation and growth control (Lv, 2012).

Core histone modifications are known to play an essential role in the regulation of chromatin organization and transcription. These modifications include acetylation, methylation, phosphorylation, ubiquitination, sumoylation and poly(ADP-ribosyl)ation. Histone acetylation is one of the best-studied modifications and is thought to be involved in both the initiation and elongation steps of transcription. The acetylation of the core histone tails alters the folding dynamics of nucleosomal arrays and 30-nm chromatin fibers and recruits specific chromatin remodeling complexes that exert the specific function(s) of chromatin (Lv, 2012).

The acetylation of histones is regulated by two highly conserved classes of histone enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which catalyze the addition and removal, respectively, of acetyl groups on histone lysine residues. Reversible histone acetylation and deacetylation are highly regulated processes that are crucial for chromatin reorganization and the regulation of gene transcription in response to extracellular conditions. The balance between the acetylation and deacetylation of histones serves as a key regulatory mechanism for gene expression and governs numerous developmental processes and disease states (Lv, 2012).

HDACs have been classified into four subfamilies based on their homologs and functional similarities (Witt, 2009). Hdac3 is a class HDAC that shares homology with yeast Rpd3. This protein is reportedly present in the nuclear, cytoplasmic and membrane fractions. The knockout of Hdac3 in mice leads to embryonic lethality before day 9.5 (Bhaskara, 2008). The inactivation of Hdac3 has been shown to delay cell cycle progression and result in cell cycle-dependent DNA damage, inefficient repair and increased apoptosis in mouse embryonic fibroblasts (Montgomery, 2008; Knutson, 2008; Zampetaki, 2010). Hdac3 has also been shown to be up-regulated in various tumor types. However, the precise function and underlying molecular mechanism of Hdac3 in these processes remain largely unknown (Lv, 2012).

The Drosophila ortholog to human Hdac3 is known to be Hdac3 or dHDAC3 (Johnson, 1998). This study used Drosophila to investigate the function of Hdac3 during development. Depletion of Hdac3 in Drosophila results in a reduction in both organ and body sizes. Hdac3 controls growth through the regulation of H4K16 deacetylation. Alterations in H4K16ac through the ectopic expression of MOF, a histone acetyltransferase that specifically targets H4K16, result in changes of cell/body size. It was also found that H4K16ac is modulated by PI3K signaling. Increasing the level of H4K16ac by depleting Hdac3 effectively reverses the PI3K-induced tissue overgrowth and alterations in the transcription profile (Lv, 2012).

Hdac3 is a component of the nuclear receptor co-repressor complex containing N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), both of which are recruited by nuclear hormone receptors to regulate gene transcription). Several substrates were found to be targets of Hdac3, including histones (Bhaskara, 2010; Johnson, 2002) and non-histone proteins (Yang, 1997; Zampetaki, 2010). Among the targets affected by Hdac3, this study found that H4K16ac is a critical epigenetic modification associated with animal growth, as demonstrated not only by the finding that alterations in H4K16ac were closely associated with Hdac3-induced organ/body growth but also by the finding that mutating H4K16 directly affected Hdac3-induced growth. Furthermore, transgenic lines in which MOF, the specific histone H4K16 HAT, was over-expressed or depleted exhibited similar changes in cell/body size, thus confirming that H4K16ac plays an essential role in animal growth. Histone H4K16 acetylation is known to function as a dual switch for higher-order chromatin and protein-histone interactions and has been shown to regulate embryonic stem cell self-renewal and cellular life span. Recent work in has suggested that H4K16ac in Drosophila not only is critical for the acetylation of H4K5, H4K8 and H3K9, which are hallmarks of active chromatin, but also exerts an effect on H3K9 methylation and the association of HP1 with chromatin, which are hallmarks of heterochromatin. It is therefore presumed that the changes in H4K16ac affect higher-order chromatin and alter the transcription of genes related to growth. However, the exact mechanism by which H4K16ac regulates the transcription of genes related to growth needs to be further investigated (Lv, 2012).

One of the main findings in this work is the genetic interaction between Hdac3/H4K16ac and the PI3K pathway. The PI3K pathway is a highly conserved signal transduction cascade from flies to humans. Previous studies have identified a number of the components of this signaling pathway. However, the mechanisms by which this pathway regulates nuclear events, such as gene transcription, remain largely unknown. This work shows that PI3K signaling modulates the acetylation of H4K16. This finding was supported by results showing that the activation of PI3K caused a corresponding reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. Furthermore, the introduction of the H4K16A mutant, in which H4K16 cannot be acetylated, further enlarged the PI3K-induced increase in ommatidial size, confirming the function of histone H4K16ac in PI3K signaling (Lv, 2012).

Although the exact mechanism by which PI3K regulates H4K16ac is still unknown, this study demonstrates that the loss of Hdac3 inhibited PI3K-mediated overgrowth, thus suggesting that PI3K targets the activity of Hdac3 and subsequently affects H4K16ac. This hypothesis is supported by the observations that Drosophila Hdac3 can form a complex with Akt and that the complex of human Hdac3 with the deacetylase activation domain (DAD), the human SMRT co-repressor and inositol tetraphosphate is required for the activation of Hdac3 enzymatic functionality (Watson, 2012). The observation that the depletion of Hdac3 decreased the level of phospho-Akt and affected the subcellular localization of GFP-PH also supported this possibility. However, the observation that Hdac3 depletion failed to counteract the PI3K-induced hyperphosphorylation of Akt while completely rescuing the decrease in H4K16ac and the tissue overgrowth induced by the PI3K over-expression indicated that Hdac3 likely counteracts the PI3K-induced tissue overgrowth by modulating the level of H4K16ac (Lv, 2012).

The hyperactivation of the PI3K pathway is known to be associated with many types of human cancer. A number of HDAC inhibitors have been developed and applied in clinical trials to inhibit tumor growth. However, the molecular mechanisms of these HDAC inhibitors in cancer prevention remain to be elucidated. The present study found that the over-expression of PI3K decreases H4K16ac in vivo. Further studies have shown that increasing the level of H4K16ac by depleting Hdac3 can antagonize the PI3K-induced tissue overgrowth. This finding, therefore, may provide further insight into the mechanisms by which the HDAC inhibitors inhibit tumor growth (Lv, 2012).

Role of histone deacetylases in gene regulation at nuclear lamina

Theoretical models suggest that gene silencing at the nuclear periphery may involve 'closing' of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). This study provides experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2 cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. The data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development (Milon, 2012).

This study provides direct experimental evidence for the long-persisting assumptions that HDACs are involved in gene silencing at the nuclear lamina, by identifying Class I enzymes HDAC1 and HDAC3 as the major players in this mechanism. Likewise gene silencing, histone hypoacetylation and chromatin compaction in the multigenic chromatin domain are lamin-dependent. Moreover, HDAC1 was identified as the key factor required for silencing and specifically responsible for histone H4 deacetylation, and the data implicate HDAC3 as an auxiliary factor specifically responsible for localization of the repressed chromatin at the lamina. The 'closed' chromatin configuration of the repressed domain also depends on HDAC1 and thus probably mediates the major repressive action of this enzyme at the nuclear periphery. Published data indicate that the 60D1 cluster interacts with HDAC1, in particular in the Crtp and Pros28.1B regions (Filion, 2010), supporting direct involvement of this enzyme in histone deacetylation. A model is proposed in which Class I HDACs participate in lamina-dependent gene silencing through diverse pathways: HDAC1, tethered to the lamin scaffold by LEM domain proteins, is involved in deacetylation of histones H3 and H4 and 'closing' of lamina-bound chromatin while HDAC3 contributes to histone H3 deacetylation and retention of the repressed chromatin at the lamina. Interestingly, a recent study showed that HDAC3 is also involved in peripheral localization of the lamina-interacting chromatin in mammals (Zullo, 2012) indicating that this mechanism is conserved between diverse animals (Milon, 2012).

Lamina-associated chromatin domains harbor numerous cell type-specific genes that must be precisely regulated to orchestrate cell differentiation and development. Genetic defects in the lamina components result in severe and currently incurable tissue degenerative disorders known as laminopathies. Identification of the key role of Class I HDACs, and particularly HDAC1, in lamina-associated gene silencing implies that modulation of this enzyme may help to restore gene expression disrupted by nuclear lamina defects, and may be instrumental in establishing new expression patterns in pluripotent cells to guide their differentiation (Milon, 2012).

DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina

A large fraction of the mammalian genome is organized into inactive chromosomal domains along the nuclear lamina. The mechanism by which these lamina associated domains (LADs) are established remains to be elucidated. Using genomic repositioning assays, this study shows that LADs, spanning the developmentally regulated IgH and Cyp3a loci contain discrete DNA regions that associate chromatin with the nuclear lamina and repress gene activity in fibroblasts. Lamina interaction is established during mitosis and likely involves the localized recruitment of Lamin B during late anaphase. Fine-scale mapping of LADs reveals numerous lamina-associating sequences (LASs), which are enriched for a GAGA motif. This repeated motif directs lamina association and is bound by the transcriptional repressor cKrox, in a complex with HDAC3 and Lap2β. Knockdown of cKrox or HDAC3 results in dissociation of LASs/LADs from the nuclear lamina. These results reveal a mechanism that couples nuclear compartmentalization of chromatin domains with the control of gene activity (Zullo, 2012).

RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock

Transcription regulation of the Drosophila hsp70 gene is a complex process that involves regulation of multiple steps including establishment of paused Pol II and release of Pol II into elongation upon heat shock activation. While the major players involved in regulation of gene expression have been studied in detail, additional factors involved in this process continue to be discovered. To identify factors involved in hsp70 expression, a screen was developed that capitalizes on a visual assessment of heat shock activation using a hsp70-beta galactosidase reporter and publicly available RNAi fly lines to deplete candidate proteins. The screen was validated by showing that depletion of HSF, CycT, Cdk9, Nurf 301, or ELL prevented full induction of hsp70 by heat shock. The screen also identified the histone deacetylase HDAC3 and its associated protein SMRTER as positive regulators of hsp70 activation. Additionally it was shown that HDAC3 and SMRTER contribute to hsp70 gene expression at a step subsequent to HSF-mediated activation and release of the paused Pol II that resides at the promoter prior to heat shock induction (Achary, 2014).

Drosophila histone deacetylase-3 controls imaginal disc size through suppression of apoptosis

Histone deacetylases (HDACs) execute biological regulation through post-translational modification of chromatin and other cellular substrates. In humans, there are eleven HDACs, organized into three distinct subfamilies. This large number of HDACs raises questions about functional overlap and division of labor among paralogs. In vivo roles are simpler to address in Drosophila, where there are only five HDAC family members and only two are implicated in transcriptional control. Of these two, HDAC1 has been characterized genetically, but its most closely related paralog, HDAC3, has not. This study describes the isolation and phenotypic characterization of hdac3 mutations. Both hdac3 and hdac1 mutations were found to be dominant suppressors of position effect variegation, suggesting functional overlap in heterochromatin regulation. However, all five hdac3 loss-of-function alleles are recessive lethal during larval/pupal stages, indicating that HDAC3 is essential on its own for Drosophila development. The mutant larvae display small imaginal discs, which result from abnormally elevated levels of apoptosis. This cell death occurs as a cell-autonomous response to HDAC3 loss and is accompanied by increased expression of the pro-apoptotic gene, hid. In contrast, although HDAC1 mutants also display small imaginal discs, this appears to result from reduced proliferation rather than from elevated apoptosis. The connection between HDAC loss and apoptosis is important since HDAC inhibitors show anticancer activities in animal models through mechanisms involving apoptotic induction. However, the specific HDACs implicated in tumor cell killing have not been identified. These results indicate that protein deacetylation by HDAC3 plays a key role in suppression of apoptosis in Drosophila imaginal tissue (Zhu, 2008).

Histone deacetylases (HDACs) are members of an ancient enzyme family that reverses acetylation of protein substrates. The most well-characterized HDAC substrates are the N-terminal tails of the histones. Acetylation of histone tail lysines generally correlates with gene activity, whereas HDAC-sponsored removal of these tail modifications frequently accompanies gene silencing. Histone acetylation state can impact gene expression through recruitment of transcriptional regulatory complexes, such as the SWI/SNF remodelling complex. Changes in charge density resulting from histone acetylation/deacetylation may also affect packaging of nucleosome arrays into higher-order arrangements that can impact transcription rates. A major HDAC regulatory function, then, is to promote gene silencing (Zhu, 2008).

The histone deacetylase HDAC1 has been the most throughly studied HDAC at the biochemical and functional levels. Extensive analysis of HDAC1 in yeast, also known as RPD3, indicates that it can deacetylate all four core histones, that it targets hundreds of genes around the genome, and confirms its major role as a direct transcriptional repressor. Biochemical studies show that HDAC1 is typically assembled into nuclear complexes, such as the SIN3 and NURD complexes. These co-repressor complexes are recruited to target genes through interactions with DNA-binding proteins. A prime example is provided by nuclear hormone receptors such as thyroid hormone receptor; the unliganded receptor recognizes target genes through its zinc finger DNA-binding domain, and it recruits a SIN3/NCoR/HDAC1 complex, which deacetylates target chromatin and leads to gene silencin (Zhu, 2008).

As a consequence of their roles with many co-repressors, HDACs have widespread function around the genome and they participate in many gene regulatory systems. In addition to steroid hormone receptor control in vertebrates and invertebrates, HDACs also function in the TGF-β pathway through Smad-Ski silencing and in repression of neuronal genes in non-neuronal tissues. In the Drosophila system, HDAC1 controls segmentation genes through interaction with the Groucho co-represso, executes Notch signalling readouts through interaction with CSL transcription factors, and HDAC1 has also been linked to silencing by Polycomb repressors. Thus, many endocrine, homeostatic, and developmental pathways employ HDACs in their gene control mechanisms (Zhu, 2008).

There are 11 HDAC family members in humans, defined by an approximately 350 amino acid homology region that encompasses the catalytic domain. These have been classified into three major subfamilies, with class I containing HDACs 1, 2, 3 and 8, class II containing HDACs 4, 5, 6, 7, 9 and 10, and HDAC11 comprising a third distinct subtype. In addition, the sirtuins represent yet another HDAC family, which are distinguished by their NAD-dependent reaction mechanism and are structurally unrelated to the family of 11 human HDACs. The large number of HDACs makes it difficult to determine which functions are shared and which can be uniquely assigned to individual family members. HDAC functional diversity is further complicated by the ability of HDACs to modify many protein substrates besides histones. Indeed, all three major HDAC subtypes are present in bacterial species, indicating that they likely evolved as protein deacetylases that only later acquired ability to act upon histones. In agreement with diversity of function, HDAC1 and 2 are largely nuclear, HDAC6 is cytoplasmic, and still other HDACs, including HDAC3, are found in both nucleus and cytoplasm. Within the nucleus, several transcription factors, including p53, GATA-1, and YY1, are HDAC substrates. In the cytoplasmic compartment, tubulin deacetylation by HDAC6 has been described. The large number of HDAC family members and the diversity of their protein substrates predict a myriad of HDAC regulatory functions in vivo (Zhu, 2008 and references therein).

HDAC functions are simpler to dissect in the Drosophila system, where there are only five HDAC family members. In addition, there are only two HDACs of the class I subtype: HDAC1 (also called DmRpd3) corresponding to the nuclear HDAC1/2 of mammals, and its most closely related fly paralog, HDAC3. Furthermore, HDAC1 and HDAC3 are the only two fly HDACs implicated in transcriptional control. Genetic studies using HDAC1 mutations have identified roles in many processes including heterochromatin silencing, segmentation, and ecdysone receptor function. However, the lack of HDAC3 mutations to date has impeded understanding of its biological functions in the fly system. This study describes isolation of HDAC3 loss-of-function alleles and presents phenotypic characterization. All five HDAC3 mutations are homozygous lethal at late larval or pupal stages. The mutant larvae have abnormally small imaginal discs, which is attributed to cell-autonomous induction of apoptosis rather than defects in cell proliferation (Zhu, 2008).

A recent transcription profile microarray study using cultured fly cells suggests that HDAC1 and HDAC3 are the only two fly HDACs with major functions in transcriptional control (Foglietti, 2006). Thus, HDAC3, the closest fly paralog by sequence, is also likely to be the most functionally related fly family member to HDAC1. Isolation of hdac3 mutations has provided the opportunity to begin to assess this HDAC1/HDAC3 relationship in vivo. The results show that both hdac3 and hdac1 mutations can suppress PEV, indicating roles for both HDACs in heterochromatin regulation. The fact that an hdac1; hdac3 double mutant displays an enhanced effect suggests that both HDACs make significant contributions to this process. The simplest molecular explanation is that both enzymes directly deacetylate histone residues that then become methylated in heterochromatin. However, RNA interference experiments using cultured fly S2 cells suggest that HDAC1 is the predominant histone-modifying enzyme, with little unique contribution detected from HDAC3, at least in this cell type (Foglietti, 2006). Thus, HDAC3 function at heterochromatin could reflect deacetylation of either histone or non-histone substrates (Zhu, 2008).

In a developmental context, requirements for HDAC3 function distinct from HDAC1 could occur at times or in cell types that accumulate HDAC3 but not HDAC1. However, the spatial distributions of hdac3 and hdac1 mRNAs are both widespread, their temporal profiles during development are similar (Cho, 2005), and this study has not detected individual tissues or cell types where hdac3 product accumulates without hdac1. In general, hdac1 mRNA levels appear more abundant than those of hdac3 especially in the CNS. This is consistent with the report that the genome-wide transcriptional response to HDAC1 knockdown in cultured fly cells is more robust and involves a larger number of affected genes as compared to HDAC3 knockdown (Foglietti, 2006). Thus, HDAC1 may be needed for certain processes that do not require HDAC3. Indeed, both in vivo results, and fly S2 cell studies (Foglietti, 2006), support a preferential role for HDAC1, as opposed to HDAC3, in controlling cell proliferation (Zhu, 2008).

The most significant HDAC3 function revealed by the genetic approach is in control of cell death. Since HDAC3 loss is by itself sufficient to trigger ectopic apoptosis, neither HDAC1 nor other fly HDACs can substitute for this requirement, at least in imaginal disc tissue. These results contrast with findings on apoptosis from an HDAC knockdown study using cultured fly cells. Although treatment with a broad-specificity HDAC inhibitor, trichostatin, did induce apoptosis in S2 cells, neither HDAC1 nor HDAC3 knockdown, nor the double knockdown, affected cell viability (Foglietti, 2006). One possible explanation for this discrepancy is that the degree of hdac3 loss-of-function produced by mutations in vivo is more severe than the degree achieved by RNA interference. Alternatively, the conflicting results may reflect tissue differences in the response to HDAC loss; S2 cells are derived from embryonic neuronal cells whereas the most dramatic induction of apoptosis is seen in a larval epithelial tissue, imaginal discs. Indeed, it is noted that there is little apoptotic induction in nervous system tissue of the same larvae that display robust induction in discs. Further studies will be needed to determine the mechanisms and pathways by which HDACs control apoptosis in various tissues during normal development as well as in mammalian cell and animal models for cancer (Zhu, 2008).

Dissecting the biological functions of Drosophila histone deacetylases by RNA interference and transcriptional profiling

Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones, thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members, this study used RNA interference in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1), but not of the other DHDAC family members, leads to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 leads to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing DHDAC2 leads to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth of phenotype and no significant deregulation of gene expression was observed upon silencing of DHDAC4 and DHDACX. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor trichostatin both lead to a G₂ arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation, and signal transduction were over-represented. A large number of these genes were shown to also be deregulated upon loss of the co-repressor SIN3. It is concluded that (1) DHDAC1 and -3 have distinct functions in the control of gene expression; (2) under the tested conditions, DHDAC2, -4, and X have no detectable transcriptional functions in S2 cells; (3) the anti-proliferative and transcriptional effects of trichostatin are largely recapitulated by the loss of DHDAC1 and (4) the deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3 (Foglietti, 2006).

Individual histone deacetylases in Drosophila modulate transcription of distinct genes

Lysine residues on the N-terminal tails of histones in chromatin are the primary targets of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in eukaryotes. Regulation of histone acetylation by these two classes of enzymes plays significant roles in controlling transcriptional activity in cells. Eukaryotic organisms have several different HDACs, but the biological roles of each HDAC are still not clear. To understand the physiological functions of HDACs, six different Drosophila HDACs were characterized, including Rpd3, HDAC3, HDAC4, HDAC6-S, HDAC6-L, and Sir2, by developmental expression pattern, transcriptional profiles of target genes, and sensitivity to HDAC inhibitors. It was found that each HDAC has a distinct temporal expression pattern and regulates transcription of a unique set of genes. Furthermore, differential sensitivity of HDACs to inhibitors was demonstrated. These results show that each individual HDAC plays different roles in regulating genes involved in various biological processes (Cho, 2005).

A histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity

Histone deacetylase (HDAC) inhibitors perturb the cell cycle and have great potential as anti-cancer agents, but their mechanism of action is not well established. HDACs classically function as repressors of gene expression, tethered to sequence-specific transcription factors. This study, carried out in cultured mammalian cells, reports that HDAC3 is a critical, transcription-independent regulator of mitosis. HDAC3 forms a complex with A-Kinase-Anchoring Proteins AKAP95 and HA95, which are targeted to mitotic chromosomes. Deacetylation of H3 in mitosis requires AKAP95/HA95 and HDAC3 and provides a hypoacetylated H3 tail that is the preferred substrate for Aurora B kinase. Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin. This transcription-independent pathway, involving interdependent changes in histone modification and protein association, is required for normal progression through mitosis and is an unexpected target of HDAC inhibitors, a class of drugs currently in clinical trials for treating cancer (Li, 2006).

The classic role of HDAC3 has been that of a transcriptional repressor of gene expression, as part of a complex tethered to sequence-specific transcription factors. This study reports the unexpected finding that HDAC3 has a critical, transcription-independent function in mitosis. In interphase cells, AKAP95/HA95 binds to the nuclear matrix and is less associated with HDAC3. HP1 proteins are recruited to methylated H3K9 in heterochromatin. When cells enter into mitosis, AKAP95/HA95 may target the HDAC3 complex to deacetylate H3, in a reaction that is blocked by HDAC inhibitors, and thereby provides a hypoacetylated H3 tail as substrate for Aurora B to phosphorylate on S10. Phosphorylation of S10 by Aurora B then dissociates HP1 proteins from methylated H3K9 residues on mitotic heterochromatin, which has been referred to as the 'meth-phos switch'. These interdependent changes in histone modification and protein association are required for normal progression through mitosis, perhaps by facilitating chromosome condensation, or by serving as the indicator for the mitotic checkpoint to control proper cell division (Li, 2006).

While the transcriptional effect of HDAC inhibitors on specific genes, such as p21 and other cell cycle-regulated genes, has been reported to contribute to their anti-tumor actions, especially in G1-phase arrest, their direct effects on histone acetylation levels may be equally important for the anti-tumor activity because of the important functions of histones in different cellular processes, including mitosis. It is increasingly clear that HDAC inhibition induces G2/M arrest in many human cell lines and causes mitotic defects in different cancer cell lines. This effect of HDAC inhibition is independent of ongoing gene transcription, suggesting direct effects of histone hyperacetylation on mitosis. These results indicate that the hyperacetylation of histones induced by HDAC inhibitors directly interfere with mitotic progression (Li, 2006).

Global histone acetylation is reduced during mitosis. The current studies reveal that HDAC3 and its partner proteins AKAP95 and HA95 are required for global histone deacetylation during mitosis. Of note, the most dramatic change in acetylation that occurs during mitosis is hypoacetylation of Lys 5 of H4, which matches the substrate specificity of HDAC3. Moreover, the results clearly show that HDAC3 is required for normal mitotic progression. This is consistent with a recent study in which knockdown of HDAC3, but not HDAC1 or HDAC2, increased cells in G2/M phase in human colon cancer cells. Furthermore, knockdown of HDAC3 or AKAP95/HA95 also mimics the effects of nonselective HDAC inhibition on phosphorylation of H3S10 and retention of HP1β proteins on mitotic chromosomes. Inhibition of HDAC3 is therefore likely to be the mechanism by which HDAC inhibitors induce the G2/M block in the cell cycle. The transcription independence of this effect, while unexpected, is completely consistent with a direct mitotic function of HDAC3 in the context of the novel pathway that that is reported here (Li, 2006).

Specific patterns of histone modification at gene promoters regulate transcription via a 'histone code'. Notably, the transient phosphorylation of H3S10 has been reported in the promoter region of many mammalian immediate-early genes, which are rapidly induced in response to extracellular stimuli including UV radiation, growth factors, and cytokines. On these promoters, the phosphorylation of H3S10 precedes the H3K14 acetylation, resulting in multiple modifications of H3 that facilitate gene activation. On the contrary, this study found that the phosphorylation of H3S10 by Aurora B during mitosis requires the previous deacetylation of histones by HDAC3. Thus, in contrast to the phosphorylation of H3S10 by other kinases that prefer preacetylated histone tails, the mitotic phosphorylation of H3S10 by Aurora B kinase is linked to the deacetylation of H3, specifically by HDAC3. This characteristic of Aurora B may be specific to metazoans because IPL1, the yeast homolog of Aurora kinase, phosphorylated both monoacetylated and unacetylated H3. In addition to H3S10, Aurora B also phosphorylates H3S28 and other proteins including his- tone H3 variant centromere protein A (CENP-A). In human cell systems, Aurora B also seems to prefer hypoacetylated H3 and CENP-A H3 as substrate for phosphorylation of H3S28 and CENP-A Ser7, respectively. The global hypoacetylation of H3 tail lysines in mitotic cells and their proximity to the major sites of phosphorylation by Aurora B kinase suggest that deacetylation of histone substrates may be a general preference for Aurora B function. The relative importance of specific hypoacetylated lysines for phosphorylation of specific serine residues remains to be elucidated (Li, 2006).

The specificity of Aurora B toward hypoacetylated histone substrate suggests a mechanistic link between HDAC3-dependent histone deacetylation and a transcription-independent mechanism of mitotic arrest. H3S10 phosphorylation during mitosis is characteristic of many organisms, and is dependent on Aurora B kinase, which plays a central role throughout different stage of mitosis, including chromosome condensation, alignment, and segregation, spindle assembly, and cytokinesis. The recent finding that Aurora-dependent phosphorylation of H3S10 dissociates HP1 from mitotic heterochromatin provides molecular insight into the function of Aurora B. The current findings implicate AKAP95/HA95 and HDAC3 as upstream regulators of this "meth-phos switch", and provide a molecular mechanism to explain the anti-cancer effects of HDAC inhibitors. Aurora B kinase itself is overexpressed in a large number of cancers. The finding that Aurora B is present in HDAC3 complexes and that its kinase activity is dramatically greater when the H3 tail is hypoacetylated suggests that the interdependence of Aurora B and HDAC3 may be a novel and specific target for cancer therapies that would overcome the toxicity of nonspecific HDAC inhibitors (Li, 2006).

Molecular cloning of Drosophila melanogaster cDNAs that encode a novel histone deacetylase dHDAC3

The steady-state level of histone acetylation in eukaryotes is established and maintained by multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) and affects both the structure and the function of chromatin. Histone deacetylases play a key role in the regulation of transcription, and form a highly conserved protein family in many eukaryotic species. This study describes the cloning, sequencing and genetic mapping of two histone deacetylase genes in Drosophila melanogaster: dHDAC1 is essentially identical to the previously cloned D. melanogaster d-Rpd3 gene and dHDAC3, a novel gene, is orthologous to the human and the chicken (Gallus gallus) HDAC3 genes. The predicted amino acid sequence (438 aa) of dHDAC3 shows 58.1% identity with dHDAC1/d-Rpd3, the only previously known member of the HDAC family in this organism. The map positions on polytene chromosomes for dHDAC1 and dHDAC3 were determined as 64C1-6 and 83A3-4 respectively. A search for other dHDAC3-like genes failed to find other potential paralogues in D. melanogaster, but identified significant homologies with bacterial and fungal genes encoding enzymes that metabolise acetyl groups, and with genes for other hydrolyases such as carboxypeptidase. In addition, histone deacetylase activity in D. melanogaster nuclear extracts can be inhibited by high concentrations of zinc and activated by low concentrations, which is identical to the properties of bovine carboxypeptidase A. On the basis of sequence and functional similarities, it is suggested that histone deacetylases are metal-substituted enzymes (Johnson, 1998).

Search PubMed for articles about Drosophila Hdac3

Achary, B. G., Campbell, K. M., Co, I. S. and Gilmour, D. S. (2014). RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock. Biochim Biophys Acta 1839(5): 355-363. PubMed ID: 24607507

Bhaskara, S., et al. (2010). Hdac3 is essential for the maintenance of chromatin structure and genome stability. Cancer Cell. 18 436-447. PubMed ID: 21075309

Cho, Y., Griswold, A., Campbell, C. and Min, K. T. (2005). Individual histone deacetylases in Drosophila modulate transcription of distinct genes. Genomics 86(5): 606-17. PubMed ID: 16137856

Filion, G. J., van Bemmel, J. G., Braunschweig, U., Talhout, W., Kind, J., et al. (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143: 212-224. PubMed ID: 20888037

Foglietti, C., et al. (2006). Dissecting the biological functions of Drosophila histone deacetylases by RNA interference and transcriptional profiling. J. Biol. Chem. 281(26): 17968-76. PubMed ID: 16632473

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date revised: 18 February 2024

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