Why is histone important

Histone tail clipping, which results in the loss of the first 21 amino acids of H3 will have similar effects. In contrast, neutral modifications such as histone methylation are unlikely to directly perturb chromatin structure since these modifications are small and do not alter the charge of histones. Numerous chromatin-associated factors have been shown to specifically interact with modified histones via many distinct domains Figure 1 3. There is an ever-increasing number of such proteins following the development and use of new proteomic approaches 60 , These large data sets show that there are multivalent proteins and complexes that have specific domains within them that allow the simultaneous recognition of several modifications and other nucleosomal features.

Domains binding modified histones. Examples of proteins with domains that specifically bind to modified histones as shown updated from reference Notably, there are more distinct domain types recognizing lysine methylation than any other modification, perhaps reflecting the modification's relative importance Figure 1.

Within this group of methyl-lysine binders, numerous domains can recognize the same modified histone lysine. In these cases, H3K4me3 directly recruits the chromatin-modifying enzyme.

A further example of specific methylated lysine binding is provided by the HP1 recognition of H3K9me3 — a mark associated with repressive heterochromatin. HP1 binds to H3K9me3 via its N-terminal chromodomain and this interaction is important for the overall structure of heterochromatin 68 , HP1 proteins dimerise via their C-terminal chromoshadow domains to form a bivalent chromatin binder. Interestingly, HP1 also binds to methylated H1. Since H1. Histone acetylated lysines are bound by bromodomains, which are often found in HATs and chromatin-remodelling complexes Recently, it has also been shown that PHD fingers are capable of specifically recognizing acetylated histones.

Mitogen induction leads to a rapid activation of immediate early genes such as c-jun , which involves phosphorylation of H3S10 within the gene's promoter Furthermore, studies in Drosophila melanogaster have indicated that this protein family is involved in recruiting components of the elongation complex to chromatin Histone modifications do not only function solely by providing dynamic binding platforms for various factors.

They can also function to disrupt an interaction between the histone and a chromatin factor. This simple mechanism seems to make sense because NuRD is a general transcriptional repressor and H3K4me3 is a mark of active transcription. Indeed, this very N-terminal region of H3 seems to be important in regulating these types of interaction, though the regulation is not solely via modification of K4.

The large number of possible histone modifications provides scope for the tight control of chromatin structure. Nevertheless, an extra level of complexity exists due to cross-talk between different modifications, which presumably helps to fine-tune the overall control Figure 2. This cross-talk can occur via multiple mechanisms I There may be competitive antagonism between modifications if more than one modification pathway is targeting the same site s.

This is particularly true for lysines that can be acetylated, methylated or ubiquitylated. II One modification may be dependent upon another. Importantly, this mechanism is conserved in mammals, including humans III The binding of a protein to a particular modification can be disrupted by an adjacent modification. This action has been described as a 'phospho switch'.

In order to regulate binding in this way, the modified amino acids do not necessarily have to be directly adjacent to each other. For instance, in S. IV An enzyme's activity may be affected due to modification of its substrate. In yeast, the scFpr4 proline isomerase catalyses interconversion of the H3P38 peptide bond and this activity affects the ability of the scSet2 enzyme to methylate H3K36, which is linked to the effects on gene transcription V There may be cooperation between modifications in order to efficiently recruit specific factors.

However, this stabilization of binding may be due to additional factors in a complex with PHF8 rather than a direct effect on PHF8 itself. Histone modification cross-talk. Histone modifications can positively or negatively affect other modifications.

A positive effect is indicated by an arrowhead and a negative effect is indicated by a flat head updated from reference There may also be cooperation between histone modifications and DNA methylation. Conversely, DNA methylation can inhibit protein binding to specific histone modifications.

From a chromatin point of view, eukaryotic genomes can generally be divided into two geographically distinct environments 3. The first is a relatively relaxed environment, containing most of the active genes and undergoing cyclical changes during the cell cycle.

These 'open' regions are referred to as euchromatin. In contrast, other genomic regions, such as centromeres and telomeres, are relatively compact structures containing mostly inactive genes and are refractive to cell-cycle cyclical changes. These more 'compact' regions are referred to as heterochromatin. This is clearly a simplistic view, as recent work in D. However, given that most is known about the two simple domains described above, references below will be defined to these two types of genomic domains.

Both heterochromatin and euchromatin are enriched, and indeed also depleted, of certain characteristic histone modifications. However, there appears to be no simple rules governing the localization of such modifications, and there is a high degree of overlap between different chromatin regions. Nevertheless, there are regions of demarcation between heterochromatin and euchromatin. These 'boundary elements' are bound by specific factors such as CTCF that play a role in maintaining the boundary between distinct chromatin 'types' Without such factors, heterochromatin would encroach into and silence the euchromatic regions of the genome.

Boundary elements are enriched for certain modifications such as H3K9me1 and are devoid of others such as histone acetylation Furthermore, a specific histone variant, H2A.

Z, is highly enriched at these sites How all of these factors work together in order to maintain these boundaries is far from clear, but their importance is undeniable. Although generally repressive and devoid of histone acetylations, over the last few years it has become evident that not all heterochromatin is the same. Indeed, in multicellular organisms, two distinct heterochromatic environments have been defined: a facultative and b constitutive heterochromatin.

A classic example of this type of heterochromatin is the inactive X-chromosome present within mammalian female cells, which is heavily marked by H3K27me3 and the Polycomb repressor complexes PRCs Indeed, recent elegant work has shed light on how H3K27me3 and PRC2 are involved in positionally maintaining facultative heterochromatin through DNA replication In this way, the histone mark is 'replicated' onto the newly deposited histones and the facultative heterochromatin is maintained.

As DNA replication proceeds, there is a redistribution of the existing modified histones bearing H3K9me3 , as well as the deposition of newly synthesized histones into the replicated chromatin. Since HP1 binds to SUV39, it is tempting to speculate that the proteins generate a feedback loop capable of maintaining heterochromatin positioning following DNA replication Furthermore, this positive feedback mechanism helps to explain, at least in part, the highly dynamic nature of heterochromatin, not least its ability to encroach into euchromatic regions unless it is checked from doing so.

In stark contrast to heterochromatin, euchromatin is a far more relaxed environment containing active genes. However, as with heterochromatin, not all euchromatin is the same.

Certain regions are enriched with certain histone modifications, whereas other regions seem relatively devoid of modifications. In general, modification-rich 'islands' exist, which tend to be the regions that regulate transcription or are the sites of active transcription For instance, active transcriptional enhancers contain relatively high levels of H3K4me1, a reliable predictive feature However, active genes themselves possess a high enrichment of H3K4me3, which marks the transcriptional start site TSS 86 , In addition, H3K36me3 is highly enriched throughout the entire transcribed region The mechanisms by which H3K4me1 is laid down at enhancers is unknown, but work in yeast has provided mechanistic detail into how the H3K4 and H3K36 methyltransferases are recruited to genes, which in turn helps to explain the distinct distribution patterns of these two modifications Figure 3.

Thus, the two enzymes are recruited to genes via interactions with distinct forms of RNAPII, and it is therefore the location of the different forms of RNAPII that defines where the modifications are laid down reviewed in reference 3. Interplay of factors at an active gene in yeast adapted from references and 3.

Taken together, we are beginning to understand how some enzymes are recruited to specific locations, but our knowledge is far from complete. In addition, another question that needs to be considered relates to how different histone modifications integrate in order to regulate DNA processes such as transcription. Thus, methylation at H3K4 is intricately linked to acetylation at H3K This is important because it prevents cryptic initiation of transcription within coding regions 95 , Together, these examples show how the recruitment of two opposing enzyme activities HATs and HDACs is important at active genes in yeast.

However, it is not clear whether these mechanisms are completely conserved in mammals. In mammals, regulatory mechanisms governing the activity of certain genes can involve specific components more commonly associated with heterochromatic events. Thus, the repressed cyclin E gene promoter appears to adopt a localized structure reminiscent of constitutive heterochromatin, i. However, unlike true heterochromatin, this is a transitory structure that is lost as the cell progresses from G1- into S-phase when the cyclin E gene is activated.

Thus, components of heterochromatin are utilized in a euchromatic environment to regulate gene activity.

Crudely speaking, full-blown cancer may be described as having progressed through two stages, initiation and progression. As we discuss below, changes in 'epigenetic modifications' can be linked to both of these stages. However, before describing specific examples, we will consider the mechanisms by which aberrant histone modification profiles, or indeed the dysregulated activity of the associated enzymes, may actually give rise to cancer.

Although it is beyond the scope of this review to fully discuss all of these possibilities, we will provide a few relevant examples highlighting these mechanisms. Mouse models are invaluable tools for determining whether a particular factor is capable of inducing or initiating tumourigenesis. When the MOZ-TIF2 fusion was transduced into normal committed murine haematopoietic progenitor cells, which lack self-renewal capacity, the fusion conferred the ability to self-renew in vitro and resulted in AML in vivo Thus, the fusion protein induces properties typical of leukaemic stem cells.

Interestingly, the intrinsic HAT activity of MOZ is required for neither self-renewal nor leukaemic transformation, but its nucleosome-binding motif is essential for both , Thus, it seems that both self-renewal and leukaemic transformation involve aberrant recruitment of CBP to MOZ nucleosome-binding sites.

These findings provide a clear indication that the dysregulated function of histone modifying enzymes can be linked to the initiation stage of cancer development. An activating mutation within the non-receptor tyrosine kinase JAK2 is believed to be a cancer-inducing event leading to the development of several different haematological malignancies, but there were few insights into how this could occur , This antagonistic mechanism was shown to operate at the lmo2 gene, a key haematopoietic oncogene 14 , , In humans, extensive gene silencing caused by overexpression of EZH2 has been linked to the progression of multiple solid malignancies, including those of breast, bladder and prostate , , This process almost certainly involves widespread elevated levels of H3K27me3, the mark laid down by EZH2.

However, it has also recently been reported that EZH2 is inactivated in numerous myeloid malignancies, suggesting that EZH2 is a tumour suppressor protein , This is clearly at odds to the situation in solid tumours where elevated EZH2 activity is consistent with an oncogenic function. One possible explanation for this apparent dichotomy is that the levels of H3K27me3 need to be carefully regulated in order to sustain cellular homeostasis.

In other words, aberrant perturbation of the equilibrium controlling H3K27me3 in either direction may promote cancer development. In this regard, it is noteworthy that mutations in UTX an H3K27me3 demethylase have been identified in a variety of tumours , supporting the notion that H3K27me3 levels are a critical parameter for determining cellular identity. Finally, changes in histone modifications have been linked to genome instability, chromosome segregation defects and cancer.

It now seems clear that aberrant histone modification profiles are intimately linked to cancer. Crucially, however, unlike DNA mutations, changes in the epigenome associated with cancer are potentially reversible, which opens up the possibility that 'epigenetic drugs' may have a powerful impact within the treatment regimes of various cancers. Indeed, HDAC inhibitors have been found to be particularly effective in inhibiting tumour growth, promoting apoptosis and inducing differentiation reviewed in , at least in part via the reactivation of certain tumour suppressor genes.

Moreover, the Food and Drug Administration has recently approved them for therapeutic use against specific types of cancer, such as T-cell cutaneous lymphoma, and other compounds are presently in phase II and III clinical trials Other histone-modifying enzyme inhibitors, such as HMT inhibitors, are presently in the developmental phase.

But before we plunge head-first into a full discovery programme for other inhibitors, we should consider a number of important issues relevant to the development of such initiatives see for full discussion.

First, we do not fully understand how HDAC inhibitors achieve their efficacy. Do they for instance exert their effects via modulating the acetylation of histone or non-histone substrates? It is not known whether this promotes their efficacy or whether it would be therapeutically advantageous to develop inhibitors capable of targeting specific HDACs. Thus, when developing new inhibitors such as those targeting HMTs, we need to consider whether we should aim for enzyme-specific inhibitors, enzyme subfamily specific inhibitors, or similarly to the HDAC inhibitors, pan-inhibitors.

Nevertheless, the fact that these drugs are safe and the fact that they work at all, given the broad target specificity, are extremely encouraging. So the truth is that even though there is still a lot to learn about chromatin as a target, 'epigenetic' drugs clearly show great promise. We have identified many histone modifications, but their functions are just beginning to be uncovered. Certainly, there will be more modifications to discover and we will need to identify the many biological functions they regulate.

Perhaps most importantly, there are three areas of sketchy knowledge that need to be embellished in the future. The first is the delivery and control of histone modifications by RNA. There is an emerging model that short and long RNAs can regulate the precise positioning of modifications and they can do so by interacting with the enzyme complexes that lay down these marks , , , Given the huge proportion of the genome that is converted into uncharacterised RNAs , , there is little doubt that this form of regulation is far more prevalent than is currently considered.

The second emerging area of interest follows the finding that kinases receiving signals from external cues in the cytoplasm can transverse into the nucleus and modify histones 14 , This direct communication between the extracellular environment and the regulation of gene function may well be more widespread. It could involve many of the kinases that are currently thought to regulate gene expression indirectly via signalling cascades. Such direct signalling to chromatin may change many of our assumptions about kinases, as drug targets and may rationalise even more the use of chromatin-modifying enzymes as targets.

The third and perhaps the most ill-defined process that will be of interest is that of epigenetic inheritance and the influence of the environment on this process. We know of many biological phenomena that are inherited from mother to daughter cell, but the precise mechanism of how this happens is unclear Do histone modifications play an important role in this? The answer is yes, and as far as we know they are responsible for perpetuating these events. However, how does the epigenetic signal start off?

Is the deposition of the modifications at the right place during replication enough to explain the process? Or is there a 'memory molecule', such an RNA, transmitted from mother to daughter cell , which can deliver histone modifications to the right place?

These are fundamental questions at the heart of 'true' epigenetic research, and they will take us a while longer to answer. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Crystal structure of the nucleosome core particle at 2. Nature ; — Histone modifications. In: Meyers R, ed. John Wiley and Sons, In Press. Parthun MR. Hat1: the emerging cellular roles of a type B histone acetyltransferase.

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Cell Mol Life Sci ; 66 — Protein arginine methylation in mammals: who, what, and why. Mol Cell ; 33 :1— Lan F, Shi Y. Epigenetic regulation: methylation of histone and non-histone proteins. We observed that the epidermis localized away from the wounded site responded to the trauma by changing the acetylation profile of histones Fig. We observed that H4K5 underwent continuous deacetylation during healing.

H4K12, however, became hyperacetylated at day 9. These findings indicate that tissue trauma triggers distant H4K12 acetylation and, consequently, gene transcription. Similar to the normal epithelium, the acetylation levels of histones H4K5 and H4K8 decreased during the healing of the skin Fig. Interestingly, H4K12 became hyperacetylated at the adjacent epithelium and the wound reepithelialization Fig. Epidermis covering the wound expressed hyperacetylation of H4K16 Fig. Changes in histone H4 acetylation observed in epidermal healing overtime.

The three graphics show the time course for the change in the acetylation pattern of histone H4 at lysines 5, 8, 12, and 16 during the wound healing. Hyperacetylation of H4K12 is found at the distant epithelium upon complete wound closure reepithelialization at day 9. Conversely, significant hyperacetylation of H4K12 occurs at day 4 and day 9. The migratory epithelial tongue displays significant deacetylation of H4K5 on day 9.

The same expression pattern was observed for H4K8. The acetylation of H4K12 was unaltered upon wound closure; however, the epithelialized wound presented increased acetylation of H4K Against initial predictions, the human genome is far smaller than anticipated suggesting that the maintenance of a complex multicellular organism requires additional regulatory machinery like histone modifications.

Histones pack the chromatin in an organized way that allows unwinding of the DNA and fast gene transcription [ 23 ]. Histone modifications include acetylation, methylation, and phosphorylation. It is perceived that histone methylation results in stable modifications, while acetylation and phosphorylation of the histones can result in a transient modification [ 24 ]. Histone methylation is also often associated with reduced gene expression.

For example, histone H3 lysine 27 trimethylation H3K27me3 that has repressor activity is found downregulated during wound healing [ 25 ]. Along with the demethylation of H3K27me3, the process of cutaneous healing also displays the acetylation of H3K9 [ 26 ]. There is a limited number of studies that explore the role of histones during wound healing [ 25 , 27 ]. Here, we decided to characterize the acetylation pattern of 4 different lysines from histone H4 during three distinct phases of healing.

We observed that histone acetylation does rapidly change its acetylation pattern during the different phases of healing. Perhaps one of the most unexpected and exciting results from this study is the modifications to the acetylation profile of histone H4 observed distant from the wound boundaries.

Our findings point towards an epigenetic mechanism capable of modifying gene expression on the healthy area around the injury. Most exciting is that we have identified H4K12 as the primary lysine hyperacetylated at distant areas from the wound and potentially a marker to identify and characterize the distant effects of wounds.

On the other hand, histone H4 lys5 H4K5 maintained similar levels of acetylation between normal and adjacent tissues to the wound by day 4. This finding suggests that H4K5 is potentially associated with the suppression of genes transcription associated with differentiation, and consequent release of epithelial migration.

The acetylation of lys16 at histone H4 plays an important role in changing the conformation of the chromatin resulting in increased transcription though the reduced inter-nucleosome interaction and consequently increased chromatin accessibility to non-histone proteins [ 28 ].

We observed that histone H4K16 is the only lysine found hyperacetylated at the closed wound area. Interestingly that upon closure, the epidermis recovering the wound site undergoes a process of thickening suggestive of intense cellular differentiation, as observed in Fig.

Cellular differentiation, as cellular senescence are essential protective mechanisms involved in reduced cellular proliferation and the maintenance of tissue homeostasis. In cancer, however, H4K16 is found hypoacetylated suggesting the disruption of cellular differentiation mechanisms and the potential in controlling tumor suppressor genes [ 29 , 30 ]. Furthermore, the hypoacetylation of H4K16 is associated with dysfunctional DNA repair mechanisms in the animal model for Hutchinson Gilford progeria syndrome that result in the activations of an accelerated aging phenotype [ 31 ].

Combined, elevated genomic instability driven by the hypoacetylation of H4K16 can either trigger accelerated senescence in normal tissues or can lead to tumor progression driven by a dysfunctional DNA repair machinery. Similar to H4K16, we have identified a spike on H4K12 acetylation during epithelial migration and proliferation at day 4 after injury.

At the same time, all other lysines from histone H4 were deacetylated compared to healthy tissues. There is little information available on the role of lysine 12 acetylation of histone H4, especially in wound healing and epithelial biology.

Acetylation of histone H4K12 along with H3K9 is associated with the maintenance of progenitor-specific characteristics of rod photoreceptor cells from the retina [ 32 ]. The maintenance of cycling progenitor cells was also associated with the inhibition of histone deacetylase 1 HDAC1 that further prevented rod cells from undergoing differentiation.

These results are somewhat aligned with our epidermal data in which migrating epithelial cells expressing high levels of H4K12 proliferate but do not undergo differentiation, as observed in the migratory epithelial tongue Fig.

From a cancer perspective, the hyperacetylation of histone H4K12 is associated with poor prognosis of pancreatic cancers [ 33 ] that, along with the rod cells data and our wound-healing results, may be correlated with the presence of undifferentiated tumor cells and reduced cellular differentiation.

The mechanism involved in tissue repair upon an injury is comprised of a multi-step process that involves the mobilization of epithelial cells, the activation of an inflammatory response, and the remodeling of the wound bed. The wound-healing process is far from simple and is likely influenced by epigenetic modifications. Our current study points towards the presence of a histone code for epidermal repair, in which each healing phase presents a dominant histone H4 hyperacetylation site.

These findings further facilitate the identification of epigenetic-controlled genes from each healing timepoint, and the testing of epigenetic drugs to treat skin wounds.

Wound repair and regeneration. Genome Biol. Article Google Scholar. The language of covalent histone modifications. Turner BM. Histone acetylation and an epigenetic code. Stillman B. Histone modifications: insights into their influence on gene expression. Tessarz P, Kouzarides T.

Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Genomic characterization reveals a simple histone H4 acetylation code. Single-cell transcriptomics of traced epidermal and hair follicle stem cells reveals rapid adaptations during wound healing. Cell Rep. Google Scholar. Gene expression profiling of cutaneous wound healing.

J Transl Med. Temporal expression of wound healing-related genes in skin burn injury. Leg Med. Deep sequencing transcriptome analysis of murine wound healing: effects of a multicomponent, multitarget natural product therapy-Tr Front Mol Biosci. Rac1 is required for epithelial stem cell function during dermal and oral mucosal wound healing but not for tissue homeostasis in mice.

Splinting strategies to overcome confounding wound contraction in experimental animal models. Adv Wound Care. The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues. Topical delivery of mTOR inhibitor halts scarring. J Dermatol Sci. Scudamore CL. A practical guide to the histology of the mouse.

Hoboken: Wiley; Cremer T, Cremer C. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet. Henikoff S. Nucleosome destabilization in the epigenetic regulation of gene expression.

Papkov VG. Functional morphology of the hypothalamus in cardiovascular pathology. Sov Med. Exploring the role of stem cells in cutaneous wound healing. These findings indicate that H4 acetylation modulates higher order chromatin structure to facilitate the histone-to-protamine transition. The depletion of either Epc1 or Tip60 perturbs histone hyperacetylation, especially H4 acetylation, and affects histone replacement during spermiogenesis Dong et al.

Germ cell-specific Sirt1 knockout mice display reduced male fertility due to decreased spermatozoa number and increased proportion of abnormal spermatozoa Bell et al.

In Sirt1 -null elongating and elongated spermatids, acetylation levels of H4K5, H4K8 and H4K12 are decreased and TP2 could not co-localize in the nucleus, leading to a chromatin condensation defect in Sirt1 -null spermatozoa Bell et al.

Thus, SIRT1 may modulate other factors to promote H4 acetylation and the histone-to-protamine transition. Figure 2 The key factors related to the histone-to-protamine transition. Acetylation at critical lysines further destabilizes the nucleosomes, while tail acetylation generates a platform for the recruitment of BRDT. Evicted acetylated histones would then be recognized by PA and degraded by proteasomes during spermatogenesis.

RNF8 could catalyze the ubiquitination of H2A. The histone acetylation might be recognized by some chromatin remodelers to confer downstream signaling, and the double bromodomain and extra-terminal domain BET proteins have been identified to be critical epigenetic readers binding to acetylated histones and modulating changes in chromatin structure and organization during spermiogenesis Berkovits and Wolgemuth, BRDT is a testis-specific BET member protein, which is expressed specifically in spermatocytes and spermatids, and contains two bromodomains that specifically recognize acetylated lysine residues Shang et al.

Remodeling assays have shown BRDT regulated the chromatin reorganization dependent acetylation in round spermatids Dhar et al. In mice, the disruption of the first bromodomain in BRDT resulted in male sterility by producing the morphologically abnormal spermatids Shang et al.

In elongating spermatids with BRDT containing a knockout of bromodomain 1 BD1 , TPs and protamines remained in the cytoplasm and histone replacement did not occur, suggesting BRDT is required for the histone-to-protamine transition by mediating the replacement of acetylated histones Figure 2 Gaucher et al.

Proteasomes catalyze ATP- and polyubiquitin-dependent protein degradation, and they are made up of a 20S catalytic core particle CP and regulatory particle RP. PA is highly expressed in the testis, and the disruption of PA results in male infertility and severe defects in spermatogenesis Ustrell et al.

During spermiogenesis, PA regulatory could directly recognize acetylated histones through a bromodomain-like module and promote their ubiquitin-independent degradation. In Pa -null spermatids, results showed that H2B, H3 and elevated H4K16ac could be detected at the end of the elongation stage Qian et al. Thus, PA specifically recognizes acetylated histones and mediates the core histones for acetylation dependent degradation through proteasomes during spermatogenesis Figure 2.

Ubiquitin is a 76 amino acid protein that is attached to target proteins to regulate several cellular processes, such as protein degradation, cell signaling, autophagy, DNA damage responses and so on Hershko and Ciechanover, ; Pickart, ; Welchman et al. The disruption of Rnf8 causes significant late-stage developmental defects in spermatids due to problematic histone-to-protamine replacement, with the canonical histones being detectable in Rnf8 -deficient mature spermatozoa Lu et al.

Further studies showed that ubiquitinated H2A and H2B were essential for the efficient recruitment of the MOF males absent on the first acetyltransferase complex, which is highly expressed in elongating spermatids and responsible for H4K16 acetylation in the chromatin Akhtar and Becker, ; Lu et al. Thus, RNF8 catalyzed histone ubiquitination could modulate H4K16ac by regulating the localization of MOF on the chromatin and facilitate histone removal in the elongating spermatids.

In mice, Miwi , Mili , and Miwi2 , the Piwi paralogs, have been identified in the testis and are required for male fertility Deng and Lin, ; Kuramochi-Miyagawa et al. In both humans and mice, mutations in the conserved destruction box D-box of HIWI and MIWI proteins, which lead to their stabilization, cause male infertility due to impaired histone ubiquitination and histone-to-protamine transition Gou et al.

The depletion of L3mbtl2 in germ cells affected male fertility by producing abnormal spermatozoa and the decrease of sperm counts. L3mbtl2 deficiency also caused the reduction of in levels of the RNF8 and histone ubiquitination in elongating spermatids, which further influenced the PRM1 deposition and chromatin condensation during spermiogenesis Meng et al.

PHF7 is specifically located in the elongating spermatid nuclei, and the disruption of Phf7 led to male mouse infertility as reduction of sperm count and the increased proportion of abnormal spermatozoa Wang et al. In Phf7 -null spermatids, the H2A ubiquitination was dramatically decreased that resulted in the histone retention and protamine replacement defect Figure 2 Wang et al. Among them, the methylation of H3K4 and plus acetylation might help to achieve a more-open chromatin configuration, whereas H3K9 and H3K27 methylation are known to be associated with a more-repressed chromatin configuration Rathke et al.

As some histone methyltransferases and demethylases are detectable during spermiogenesis Godmann et al. Although few mouse models exist that allow precise detection of methylation activity that directly regulates histone replacement during spermiogenesis, some studies have revealed that histone methylation may modulate the histone-to-protamine transition through some other ways.

In mice, the reduction of Pygo2 influenced the Tnp, Prm genes expression and caused the abnormal nuclear condensation, which further led to male sterility Nair et al. The predominant histone methyltransferase SETD2 SET domain—containing 2 catalyzes the H3K36me3, and knocking out Setd2 in mouse germ cells causes aberrant spermiogenesis, resulting in complete male infertility.

The loss of Jhdm2a in mice exhibits post-meiotic chromatin condensation defects and leads to male infertility. Although global H3K9 methylation has no effect in Jhdm2a -null testis, JHDM2A directly binds to and controls H3K9 methylation at the promoter of Tnp1 and Prm1 genes, which further regulates the sperm genome packaging and chromatin condensation Okada et al.

Histone phosphorylation is involved in various cellular processes Rossetto et al. In mice, targeted deletion of Tssk6 leads to male sterility caused by the impairment in morphology and motility of spermatozoa Spiridonov et al. H4S1 phosphorylation is highly expressed in mouse spermatocyte, round and elongating spermatids Krishnamoorthy et al.

H4S1 phosphorylation has been found to be essential for chromatin compaction and concomitantly histone accessibility Krishnamoorthy et al. Although many core histones and histone variants phosphorylation have been identified in germ cells, their physiological roles need further investigation.

A variety of histone lysine modifications have been identified, including butyrylation, crotonylation, malonylation, propionylation, and succinylation Tan et al. Kcr Lysine crotonylation is a newly identified histone modification and is detectable in elongating spermatids, which regulated testis-specific genes activation in post-meiotic germ cells Tan et al. Accordingly, Cdyl -deficient male mice show reduced fertility, decreased epididymal sperm count and sperm cell motility, and dysregulated histone Kcr Liu et al.

In the Cdyl -deficient mouse testes, further analysis showed that the elevated TP1 and PRM2 were localized in a chromatin-free regions Liu et al. Poly-ADP-ribosylation PARsylation is a common protein PTM post-translational modification observed in higher eukaryotes and involved in many different fundamental cellular functions. Thus, PARsylation is essential for the histone-to-protamine replacement, yet the precise PARsylation histone sites need further characterization. Between histone eviction and protamine incorporation in the nuclei of spermatids, about ninety percent of the chromatin components consist of TPs, which are arginine- and lysine-rich proteins encoded by Tnp1 and Tnp2 Meistrich et al.

However, the functional roles of each TP are still controversial Rathke et al. These differences might reveal their unique roles during mammal spermiogenesis, as single knockout of either Tnp1 or Tnp2 leads to little morphological alteration of spermatozoa in mouse models. Thus, TP1 and TP2 may compensate for each other in vivo.

Indeed, Tnp1 and Tnp2 double-knockout mice show severe abnormal spermiogenesis with a general decrease in sperm motility and abnormal sperm morphology Shirley et al. The chromatin condensation is perturbed in the Tnp1 and Tnp2 double-knockout mice as severe histones retention is detectable, indicating TPs function redundantly yet have unique roles in the histone-to-protamine transition Shirley et al.

Protamines are basic proteins that replace TPs in late spermatids Rathke et al. Two protamine genes Prm1 and Prm2 localize on the same chromosome in both humans and mice Balhorn, Unlike Tnp genes, the disruption of either Prm1 or Prm2 leads to the male infertility Cho et al. Protamines have multiple PTM sites, and a total of 11 PTMs have been identified on the protamines of mouse spermatozoa, including acetylation, phosphorylation and methylation Brunner et al.

Targeted Camk4 knockout male mice are infertile, and the transition protein displacement by PRM2 is perturbed as a specific loss of PRM2 and prolonged retention of TP2 in Camk4 -null spermatids. Thus, the specific post-translational modifications on protamines may also be essential for the histone-to-protamine transition. During the histone-to-protamine transition, many epigenetic regulators work together to facilitate paternal genome re-organization and packaging into the highly condensed nuclei of spermatozoa, through histone variation, specific histone modification and their related chromatin remodelers.

Any defects during the histone-to-protamine transition would lead to male infertility Bao and Bedford, While the morphological changes during spermiogenesis are well characterized, the precise molecular mechanisms underlying the chromatin re-organization, in particular the transition from histones to protamines, are still unclear. These problems may be ascribed to a lack of experimental methods, which could fully recapitulate germ cell development in vitro.

Further physiological insights may be gained by developing an in vitro germ-cell culture system that more accurately recapitulates the in vivo histone-to-protamine transition. Many histone variants modulate histone replacement by regulating the chromatin structure; therefore, nucleosomes containing these histone variants often maintain a relatively decondensed and open chromatin configuration, facilitating histone replacement during spermiogenesis.

The redundant function of histone variants in modulating chromatin configuration ensures that defects in some histone variants have a limited effect on spermatogenesis. Indeed, some mutant histone variants in mouse models are dispensable for male fertility, and mice may show elevated levels of compensatory histones or histone variants.

However, the redundant function of histone variants makes it difficult to explore the precise role of each histone variant in histone replacement. Although many histone modifications have been identified during the histone-to-protamine transition, many studies are descriptive and correlative. The direct manipulation of histone modification sites to reveal function is still urgently needed.

The following open-ended questions still need to be answered to provide in-depth investigation in the field. In addition to the histone variants and modifications mentioned above, what other novel histone variants and modifications participate in the histone-to-protamine transition? How can we identify them? How and where do histone variants replace canonical histones? What signal is needed to initiate replacement? As histone variants and modifications are identified that participate in the histone-to-protamine transition, how do we establish an epigenetic modulating network for this process?

Which type of histone code is the initiating code? Histone hyperacetylation works as a determining event during the histone-to-protamine transition. Is this histone hyperacetylation an initial signal or an indirect consequence of prior events? Chromatin assembly is modulated by histone chaperones or other chromatin remodelers. Which histone variants or modifications send a signal to the chaperones?

How do protamines replace the transition proteins? What are the detailed functional roles of transition proteins? These questions and their underlying ideas need further investigation and refining to help us more thoroughly understand the complex molecular relationships and exact regulating mechanisms of the histone-to-protamine transition.

All authors listed have made a substantial, direct and intellectual contribution to the work and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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