Cracking the survival code

Abstract

Modifications of histones, the chief protein components of the chromatin, have emerged as critical regulators of life and death. While the “apoptotic histone code” came to light a few years ago, accumulating evidence indicates that autophagy, a cell survival pathway, is also heavily regulated by histone-modifying proteins. In this review we describe the emerging “autophagic histone code” and the role of histone modifications in the cellular life vs. death decision.

Introduction

Wrap and pack: Histones are chief protein components of chromatin

Chromatin is the polymer formed by DNA together with related histone proteins. It is organized in repeats of nucleosomes that contain an octamer of histones formed by the 4 core histones—H2A, H2B, H3, and H4, and 2 laps of, on average, around 147 DNA base pairs that wrap around the histone octamer.^1,2 (Fig. 1). The nucleosomal structure resembles “beads on a string,” where the “beads” represent the individual nucleosomes and the “string” represents the linker DNA. Linker histones such as histone H1, and other nonhistone proteins can bind to the nucleosomal arrays to compact the chromatin further, thereby contributing to the formation of higher-order chromatin structures.

Figure 1. Chromatin structure. Chromatin is made of repeating units of nucleosomes, which consist of ~147 base pairs of DNA wrapped around a histone octamer consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. Linker histone H1 is positioned on top of the nucleosome core particles stabilizing higher order chromatin structure. The histones are subject to a wide variety of posttranslational modifications, primarily on their N-terminal tails, but also in their globular core region.

Decorating histones and their tails: Histone posttranslational modifications

Like other proteins, histones are subject to posttranslational modifications (PTMs), including phosphorylation, ubiquitination, methylation, and acetylation. These modifications and the corresponding demodifications are all executed and governed by specific enzyme complexes.³ Important groups of enzymes in this histone regulatory network are the histone acetyltransferases (HATs or KATs for lysine specificity), the histone deacetylases (HDACs), histone methyltransferases (HMTs) and histone or lysine demethylases (KDMs). The most well- recognized action of a HAT is the addition of an acetyl group to lysines on the N terminus of histones, by which they mediate chromatin decondensation. HDACs substract the acetyl groups and induce a tighter binding of histones to DNA.^4,5 Histone methylation is more complex, as methylation comes in 3 steps, mono-, di- and trimethylation, and the effect of lysine methylation of a histone depends strictly on which lysine is methylated and to what extent. Histone PTMs are especially prevalent within the histone N-terminal tails, which protrude from the nucleosomal surface, but are also on the histone globular core region.^6,7 These histone PTMs have the potential to affect the overall structure of chromatin with clear functional consequences.^5,8 Histone modifications contribute to the regulation of transcription, transcriptional elongation, higher order chromatin structure, and other fundamental events. An early demonstration showed that PTMs can alter the electrostatic charge of the histone thus altering the DNA binding ability, and thereby regulating the packaging of chromatin and the access of DNA-binding proteins such as transcription factors. Although still valid, recent studies have demonstrated many other models for the effects resulting from chromatin modification. A major role for the PTMs is to function as signal platforms, or “beacons,” to recruit specific modules to local chromatin, in a context- and modification-dependent manner. Such modules may initiate or repress transcription directly, but many new studies also suggest that such modules can influence the stepwise recruitment of secondary modules and thereby transcription rate, protection of DNA, and sensitivity to signals in a more fine-tuned manner.⁹

What would histone posttranslational modifications do during autophagy?

Macroautophagy, hereafter referred to as autophagy, is a conserved, typically catabolic, process that leads to the lysosomal degradation of cytoplasmic components, abnormal protein aggregates, and damaged organelles.^10,11 Autophagy involves dynamic membrane-rearrangements controlled by a set of autophagy-related (ATG) proteins.¹² The kinase mechanistic target of rapamycin (MTOR) is a central regulator for autophagy induction, where activated MTOR suppresses autophagy, and negative regulation of MTOR promotes it. Paradoxically, while autophagy is considered to be a protective process, it can be involved in cell death as well;¹³ however, it is yet unclear what regulates this life or death decision.¹⁴ Autophagy is implicated in a number of physiological processes important for human health and disease.¹⁰ Therefore, understanding the pathways regulating the autophagic life and death decision and its cellular long-term effects might help to improve autophagy-based clinical treatments.^15,16 A role for histone PTMs in autophagy was not obvious at first; indeed until recently, nuclear regulation of autophagy was not expected as reports indicated that cells without a nucleus can still accumulate autophagic vesicles as a response to various inducers of autophagy.¹⁷ However, in recent years, compelling evidence revealed that de facto the nucleus is a major regulator of autophagy. Indeed, the autophagic process appears to encompass transcriptional and epigenetic programs, including histone PTMs. Here we will review histone PTMs that have been directly or indirectly linked to autophagy, and their functional consequence on this biological process.

Autophagy-Related Histone Modifications

First attempts, first observations, and a first hint

First attempts to link autophagy to histone modifications came from the use of HDAC inhibitors, which promote histone hyperacetylation and thereby affect gene expression patterns. In 2004, Shao and coworkers presented data indicating that the HDAC inhibitors butyrate and suberoylanilide hydroxamic acid (SAHA) can induce autophagic cell death in a range of human cancer cell lines.¹⁸ Of note, these investigations were performed before the discovery that the acetylation level of histones can regulate autophagy, which led to a focus on the interpretation of the effects of HDAC inhibitors toward nonhistone targets.^19-21 However, a first report of histone PTMs occurring upon autophagy induction, came from work by Madeo, Kroemer, and coworkers. In aging yeast, they determined that the cytoprotective induction of autophagy by spermidine relies on the inhibition of HAT activity, in particular Iki3 and Sas3, which causes global hypoacetylation of histone H3, and therefore probably reflects the repression of gene expression. In contrast, some genes, for example ATG genes, seem to be protected from this global hypoacetylation, and therefore maintain accessible promoters, allowing their selective transcription. The authors propose that this might be an important mechanism for saving resources during starvation.²² Nevertheless, to our knowledge, to identify the first hint on the potential regulation of autophagy by histone PTMs, one has to look back to the beginning of this millennium. Indeed in 2001, Yamaki and colleagues demonstrated, that yeast lacking the histone chaperone ASF1A (anti-silencing function 1A histone chaperone), undergo cell death exhibiting a plethora of autophagic bodies.²³ In yeast, acetylation of histone H3 lysine 56 (H3K56ac) is catalyzed by the HAT Rtt109, which requires Asf1 for function.²⁴

H3K56ac

Histone H3 lysine 56 is located at a rather unique position in the nucleosome; this mark localizes at the entry and exit points of a nucleosome. Noteworthy the histone-DNA interactions at these sites are weakened by H3K56 acetylation.²⁵ Furthermore, acetylation of H3K56 has a crucial role for the integration of histone variants and for packaging of DNA into chromatin following DNA replication and repair. H3K56ac also contributes to the control of gene expression.^25-28 Remarkably the use of a histone H3-H4 yeast mutant library identified specifically the H3K56ac residue as being reduced by the TOR inhibitor rapamycin. It was proposed that TOR signaling regulates H3K56ac through repression of the H3K56 HDACs Hst3 and Hst4.²⁸ In humans, both EP300/KAT3B/p300 and KAT2A/GCN5 are responsible for the acetylation of H3K56.²⁹ In fact, knockdown of EP300 can stimulate autophagy, whereas overexpression of EP300 inhibits starvation-induced autophagy.³⁰ However, the actual effect of the EP300 histone acetyltransferase activity on autophagy remains to be determined, since EP300 regulates the acetylation of various known components of the autophagy machinery, including ATG5, ATG7, MAP1LC3, and ATG12.³⁰ Furthermore, it was proposed that KAT2A can generate acetylated substrates for the autophagic pathway and/or directly promote autophagy.³¹ The mechanism regulating deacetylation of H3K56 is controversial; indeed 5 different HDACs—HDAC1, HDAC2 SIRT1, SIRT2, and SIRT3—have been described to deacetylate this residue.^8,29,32,33 Remarkably all of these HDACs are regulators of autophagy, reversing acteylation of nonhistone targets such as ATGs, and certainly also due to their action on intrinsic natural histone targets.^19,34,35 In summary, acetylation of H3K56, catalyzed by EP300 or KAT2A (Rtt109 in yeast) and displaced by various HDACs including SIRT1 to SIRT3 and HDAC1/2 (Hst3 and Hst4 in yeast), appears to be positively regulated by the MTOR-signaling pathway and repressed upon autophagy induction (Fig. 2).

Figure 2. Autophagic histone modifications. Posttranslational modifications on histone tails that occur in autophagic cells are represented. These covalent modifications are added or removed by histone modifying enzymes often termed as “writers” and “erasers.” The modifications shown are discussed in the main text. ac, acetylated; me, methylated.

H4K16ac

Unlike most histone modifications, acetylation of histone H4 lysine 16 (H4K16ac) plays a central role for the regulation of higher-order chromatin structures beyond the level of nucleosomes. In particular, H4K16ac influences the functional interactions between histones and the chromatin fiber, providing a potential regulatory mechanism for chromatin folding and playing an important role in transcription.³⁶ In multiple cell types, the induction of autophagy, by various stimuli, has been coupled to a reduction of H4K16ac. In addition, genome-wide H4K16 deacetylation is associated predominantly with the downregulation of autophagy-related genes, such as ATG9A, GABARAPL2, MAP1LC3, ULK1, ULK3, and VMP1.³⁷ Remarkably, whereas many HATs show either little substrate specificity or preference for other residues, KAT8/hMOF/MYST1 is necessary for the majority of H4K16 acetylation. KAT8’s antagonizer is the HDAC SIRT1, which targets a plethora of nonhistone proteins, but has H4K16 as its primary histone target.³⁸ It appears that KAT8 downregulation is part of the autophagy program, which in turn contributes to reduction of H4K16ac.³⁷ The observed dramatic changes in the levels of H4K16 acetylation, through the downregulation of KAT8, and associated transcriptional gene regulation suggested that there might be a functional role for this epigenetic change during autophagy. Investigations of the autophagic flux revealed that inhibition of the autophagic H4K16 deacetylation does not inhibit autophagy, but on the contrary increases autophagic flux. The observed increase in autophagic flux is in accordance with the genome-wide investigations, which provide compelling evidence that H4K16 deacetylation is directly linked to a downregulation of autophagy-related genes.³⁷ In summary, KAT8 is a substrate for autophagy, and its degradation and the consequent reduction of H4K16 acetylation, and the H4K16 deacetylation-dependent regulation of autophagy-related genes, in turn, regulates the autophagic flux.

H3K4me3

Di- and trimethylation of histone H3 at lysine 4 (H3K4me2/me3) is involved in transcriptional competence and activation. The highest H3K4me3 levels occur near the transcriptional start sites of highly expressed genes.³⁹ H3K4 is methylated by the SET1 and mixed lineage leukemia family of HMTs⁴⁰ and removed by KDM1A/LSD1 (lysine (K)-specific demethylase 1A) and KDM5/JARID1 [lysine (K)-specific demethylase 5] family of histone lysine demethylases (KDMs).⁴¹ Genome-wide investigations showed that the H4K16ac and H3K4me3 histone marks reside within single nucleosomal units in human cells.^42,43 The coexistence of H4K16ac with H3K4me3 marks is consistent with the identification of multiple molecular interactions between the enzymes that are responsible for installing these marks, KMT2A/MLL1 [lysine (K)-specific methyltransferase 2A] and KAT8.^42,44,45 In agreement with this molecular connection, autophagy also leads to a reduction in H3K4me3 levels in a wide variety of cell lines from yeast to humans. The disappearance of H3K4me3 alongside H4K16ac is likely to be involved in a general transcriptional inhibition to save energy during prolonged starvation. Interestingly, the WNT/β-catenin signaling pathway has recently been reported as a negative regulator of autophagy by inhibiting the transcription of SQSTM1/p62. During autophagy WNT is released from the SQSTM1 promoter and degraded, increasing SQSTM1 gene expression and autophagy levels.⁴⁶ Furthermore, WNT interacts with, and induces, mixed lineage leukemia that promotes H3K4me3 levels at promoters.^47,48 By blocking WNT in propagating tumor cells a strong reduction in global H3K4me3 levels is observed.⁴⁸ Hence, the degradation of WNT during autophagy is potentially involved in the downregulation of global H3K4me3 levels.

H4K20me3

H4K20 methylation is associated with transcriptional repression.⁴⁹ Hence, H4K20me3 is present in constitutive heterochromatin regions,^49,50 especially in chromosomal regions that contain silenced genes.⁵¹ The methylation of H4K20 is performed by a series of HMTs, including SETD8 and SUV420 (SUV420H1 and SUV420H2). SETD8 is responsible for the majority of the monomethylation on H4K20. H4K20me1 then acts as a substrate for the SUV4-20 enzymes, which are responsible for the methylation reactions to H4K20me2/3. PHF8 (PHD finger protein 8) has been identified as a H4K20 demethylase. Interestingly, serum starvation, a prominent inducer of autophagy, has a marked impact on the global level of H4K20me3. Thus, Kourmouli et al. described how a decrease in serum in the culture medium from 10% to 0.1% leads to a significant, serum concentration dependent, increase in H4K20me3. Furthermore, the amount of brightly stained cells increases from 10% up to 90% under serum starvation.⁵⁰ Supporting the role of H4K20me3 as a functional component of autophagy regulation is the fact that the previously described autophagy regulatory H4K16ac mark is heavily connected to H4K20me3. Recently it was suggested that H4K20me is deposited through a preceding deacetylation of H4K16ac and this process determines the levels of H4K20me3 throughout the genome.⁵² The H4K16ac-H4K20me3 connection is evident particularly in the control of RNA polymerase II (RNAP II/Pol II) pausing. This regulatory mechanism is an important regulatory step in transcription where RNA synthesis begins but “pauses” 20–50 nucleotides downstream of the transcriptional start site.⁵³ It is expected that thousands of human genes, about 20–30% of the genome, are regulated by RNAP II pausing. In this respect, H4K20me3 acts by counteracting H4K16ac, enhancing RNAP II pausing. Therefore, during autophagy an increase in H4K20me3 together with a decrease in H4K16ac can act in conjunction to inhibit the expression of a subset of genes under control of RNAP II pausing.

H3K9me2

The most recent newcomer to the field of autophagy-regulated histone modifications is the dimethylation of lysine 9 on histone H3 (H3K9me2). Recently, 2 papers described the removal of H3K9me2 from the promoters of several autophagy-related genes and how this modification can affect the outcome of autophagy.⁵⁴ H3K9 can be mono-, di- or trimethylated by a whole set of different histone methyltransferases (HMTs) such as SUV39H1 [suppressor of variegation 3-9 homolog 1 (Drosophila)] or EHMT2/G9a (euchromatic histone-lysine N-methyltransferase 2), or acetylated by HATs such as KAT2A and KAT2B/PCAF.⁵⁵ The decrease of H3K9 acetylation levels is required for an increase of H3K9me2. H3K9 can be demethylated by several members of the family of histone lysine demethylases (KDMs). Methylation of this residue is mainly associated with transcriptional repression. EHMT2 associates with the promoters of core autophagy genes including LC3B and WIPI1, and also with TP53INP2/DOR, acting as a transcriptional repressor. However, when autophagy is induced, EHMT2 leaves the promoters and allows demethylation as well as acetylation of H3K9 thereby increasing the expression levels of autophagy-related proteins.⁵⁴ Furthermore, the specific EHMT2 inhibitor BRD4770 increases the level of BNIP3 (BCL2/adenovirus E1B 19kDa interacting protein 3) and even LC3 at higher doses. Also, when TP53-mutant PANC-1 cancer cells are cotreated with the autophagy inducer gossypol, autophagy and even autophagy-related cell death are enhanced.⁵⁶ In summary, the EHMT2-mediated methylation level of H3K9 acts as a repressor of autophagy under normal conditions. However, after autophagy induction EHMT2 is readily removed from promoters of autophagy-related genes leading to disappearance of H3K9me2, increase in H3K9ac and thus transcription of genes. When autophagy is induced alongside a persistent reduction in EHMT2, autophagy seems to become overstimulated resulting in cell death with autophagic features.

Decision makers? Survival vs. death histone codes

Allis and Turner proposed the “histone code” hypothesis that encapsulates the role of histone modifications for chromatin structure and the corresponding regulation of nuclear functions.^57,58 According to this hypothesis, the combination of specific covalent posttranslational histone modifications influences the chromatin structure and leads to an impact on the transcriptional outputs.⁵⁷ This hypothesis was further developed with the concept that various combinations of histone posttranslational modifications are in control of specific chromatin-related functions and processes.⁵⁹ The recently described connections between autophagy and histone modifications make it appear likely that they are more than “useless” bystanders of the autophagic process. Specific histone modifications have also been linked to apoptotic chromatin changes, providing evidence for the existence of an apoptotic histone code.⁵ Similar to this “death code,” a set of changes in histone modifications occur during autophagy, creating an autophagy histone code, in other words a “survival code,” which determines the destiny of the cell (Fig. 2).

Autophagy is usually seen as a cellular survival process to cope with a limited supply of nutrients, damaged organelles, or stress conditions. However, its overstimulation can also lead to autophagic cell death.^13,14 Histone modifications are involved in early stages, namely the removal of H3K9me2, and late stages, namely H4K16 deacetylation, of autophagy ensuring a level of autophagy required for survival. Noteworthy, the same modifications that are involved in autophagy also show a remarkable inverse correlation with the apoptotic histone marks indicating that an interference with the autophagic histone code can push the cell toward cell death (Fig. 3). Moreover, the same histone modifications known to date to take part in the “survival histone code” or the “death histone code” are also involved in carcinogenesis, thus representing histone onco-modifications. This is rather unsurprising as deregulations of autophagy and apoptosis are both hallmarks of cancer. While the inhibition of the apoptotic program is generally beneficial for cancer cells the situation is more complicated with autophagy. In general, during early cancer development a high level of autophagy can result in cancer cell removal, whereas established tumors rely on an increased autophagy for resistance to stress, including a limited nutrient supply.¹²

Figure 3. Autophagic vs. apoptotic histone codes. Overview of autophagy-related histone modifications that are also linked to apoptosis and/or carcinogenesis. Autophagy-related histone modifications display an intriguing inverse relationship to apoptosis-related histone modifications ac, acetylated; H, histone; K, lysine; me, methylated.

Others and we have shown that antagonizing the survival marks during autophagy can indeed induce cell death.^37,56 Both removal of the HMT EHMT2 by the specific chemical inhibitor BRD4770, decreasing the level of the repressive H3K9me2 histone modification, or increase of the active H4K16ac histone modification (through overexpression of KAT8, or antagonizing SIRT1 using valproic acid, Ex527 or siRNA) during autophagy, result in cancer cell death. Inhibition of EHMT2 or induction of H4K16ac both lead to a more open chromatin structure eliminating the tight control of autophagy-related gene expression potentially resulting in an autophagic overstimulation and finally cell death.

Perspectives

Dysregulation of the histone codes

Overall it appears that histone modifications finally claim their place as central regulators of autophagy. Within the last few months, the nucleus returned to the spotlight of autophagy research^28,37,54,60 and the HATs and HDACs that were long known to be involved in the cytosolic control of autophagy seem to regulate their name-giving targets, the histones, upon autophagy induction as well. There is striking but unsurprising correlation in regulatory histone modifications during autophagy, apoptosis, and cancer development. Deregulation of both apoptosis and autophagy and also histone modifications are tightly linked to cancer. Thus, distortion of the action of a few histone-modifying enzymes can have enormous impact on disease progression, whereas their restoration or modification could be beneficial for a plethora of human conditions including cancer. There is no doubt that the histone survival code only represents the tip of the iceberg of regulatory autophagic histone modifications. While the characterization of the “death histone code” is still in its infancy, the “survival histone code” is just born. Moreover, how these histone PTMs contribute to autophagy (transcription, silencing, pausing, autophagy flux control, etc.) remains to be elucidated in detail.

Glossary

Abbreviations:

ac

acetylated

ATG

autophagy-related

EHMT2

euchromatic histone-lysine N-methyltransferase 2

H2A

histone 2A

H2B

histone 2B

H3

histone 3

H4

histone 4

HAT

histone acetyltransferase

HDAC

histone deacetylase

HMT

histone methyltransferase

K

lysine

KAT

K (lysine) acetyltransferase

KDM

histone lysine demethylase

(MAP1)LC3

microtubule-associated protein 1 light chain 3

MTOR

mechanistic target of rapamycin

me

methylated

RNAP II/Pol II

RNA polymerase II

PTM

posttranslational modification

S

serine

SAHA

suberoylanilide hydroxamic acid

SIRT

sirtuin

Suv

suppressor of variegation

WNT

wingless-type MMTV integration site family