Abstract: Although they are one of the oldest family of proteins known (first described in 1884 by Kossel), histones continue to surprise researchers with their ever expanding roles in biology. In the past 25 years, the view of core histone octamers as a simple spool around which DNA in the nucleus is wound and linker histones as mere fasteners clipping it all together has transformed into the realization that histones play a vital role in transcriptional regulation. Through posttranslational modifications, histones control the accessibility of transcription factors and a host of other proteins to multiple, conceivably thousands of, genes at once. While researchers have spent decades deciphering the role of histones in the overall structure of chromatin, it might surprise some to find that an entirely separate faction of scientists have focused on the role of histones beyond the confines of the nuclear envelope. In the past decade, there has been an accumulation of observations that suggest that histones can be found at the mitochondrion during the onset of apoptotic signaling and even at the cell surface, acting as a receptor for bacterial and viral proteins. More provocatively, immunologists are becoming convinced that they can also be found in the lumen of several tissues, acting as antimicrobial agents--critical components of an ancient innate immune system. Perhaps nowhere is this observation as dramatic as in the ability of neutrophils to entrap bacterial pathogens by casting out "nets" of DNA and histones that not only act as a physical barrier, but also display bactericidal activity. As our views regarding the role of histones inside and outside the cell evolve, some have begun to develop therapies that either utilize or target histones in the fight against cancer, microbial infection, and autoimmune disease. It is our goal here to begin the process of merging the dichotomous lives of histones both within and without the nuclear membrane.
Key words: histone, histone H1, innate immunity, antimicrobial peptides, necrosis, antibody therapeutics, autoimmune disease.
Resume : Quoiqu'elles appartiennent a une des premieres familles de proteines connues (decrites pour la premiere fois en 1884 par Kossel), les histones continuent de surprendre les chercheurs avec leurs roles de plus en plus nombreux en biologie. Au cours de 25 dernieres annees, la conception que l'on avait de l'octamere d'histones intranucleolaires (core histones) en tant que simple bobine autour de laquelle l'ADN nucleaire s 'enroule, et des histones internucleolaires en tant que simples attaches reliant le tout, s'est transformee lorsque l'on a realise que les histones jouaient un role essentiel dans la regulation de la transcription. A travers des modifications post-traductionnelles, les histones controlent l'accessibilite des facteurs de transcription et autres proteines, a plusieurs, sinon des milliers de genes a la fois. Alors que les chercheurs ont passe des decennies a decortiquer le role des histones dans la structure globale de la chromatine, il peut etre surprenant pour certains de constater qu'une faction entierement separee de chercheurs s'etait concentree sur le role que jouent les histones au-dela des confins de l'enveloppe nucleaire. Au cours de la derniere decennie, on a vu l'emergence d'observations suggerant que les histones puissent se trouver dans la mitochondrie au declanchement des signaux apoptotiques et meme a la surface de la cellule, agissant comme recepteurs de proteines virales ou bacteriennes. De maniere encore plus provocatrice, les immunologistes se sont convaincus qu'elles puissent se trouver dans le lumen de plusieurs tissus, agissant comme composante antimicrobienne essentielle du systeme immunitaire inne. Mais rien ne va aussi loin que cette observation de la capacite qu'auraient les neutrophiles de capturer les pathogenes bacteriens en tissant des filets d'ADN et d'histones qui agiraient non seulement comme barriere physique, mais exerceraient aussi une activite bactericide. Au fur et a mesure de l'evolution de notre perception du role des histones a l'interieur et a l'exterieur de la cellule, certains ont commence a developper des therapies qui utilisent ou qui ciblent les histones dans la lutte contre le cancer, les infections microbiennes et les maladies auto-immunes. Notre but ici est d'amorcer le processus d'unification des fonctions dichotomiques des histones a l'interieur ou hors de la membrane nucleaire.
Mots cles : histone, histone H1, immunite innee, peptides antimicrobiens, necrose, therapie par anticorps, maladie autoimmune.
[Traduit par la Redaction]
At home in the nucleus
In the history of molecular and cellular biology, technological advancements have brought us to a point where most mysterious phenomena can be elucidated by a group of dedicated scientists in only a matter of decades. For instance, think about how long it really took to elucidate the variety and transcriptional roles of each of the RNA polymerases. For the moment, one does not find reports about RNA polymerases suddenly having entirely new biological roles that never would have been dreamed of a decade ago. In this context, the histones have a unique place in biology. Histones are one of the oldest family of proteins known, having been discovered by Albrecht Kossel in 1884. In fact, histones are so old, the reasoning behind their given German name, histon, has been lost to obscurity (perhaps it is a derivation from the Greek histanai or histos: to set in place). Albrecht eventually won the 1910 Nobel Prize in Physiology or Medicine for his work on histones and nucleic acids, and for about a century, those interested in understanding the workings of the nucleus came to the conclusion that histones set the DNA in place.
What makes the histones unique in the annals of cell biology is the realization that, nearly 125 years after their discovery, scientists continue to rediscover histones in new and unique roles. Indeed, hardly a year passes without the discovery of yet another histone variant. Compound each of these variants with a myriad of post-translational modifications that have yet to be fully characterized (such as acetylation, methylation, poly-(ADP ribosyl)ation, ubiquination, sumoylation, and how could anyone forget, phosphorylation) and what was once a small collection of relatively highly conserved proteins becomes a rather diverse assortment of transcriptional regulators for multiple, conceivably thousands of, genes at once. Initially discovered as the consummate structural supports for DNA, for the past 25 years, chromatin researchers have been rediscovering histones as regulators of gene transcription. In this review, we wish to look beyond the gates of the nuclear garden and discuss how cell biologists and immunologists are rediscovering histones in other, more surprising roles.
A word about terminology: in much of the work we describe, both core and linker histones play a prominent role, hence, we will refer to both categories when using the term "histones;" however, on those occasions when discussion centers on only 1 category or class of histones, we will make those distinctions clear. While the core histones have a single nomenclature, the linker histones do not. In fact, much of the literature still uses the numerical nomenclature of Doenecke's laboratory (Eick et al. 1989) on some occasions and the alphabetic nomenclature of Seyedin and Kistler (Seyedin and Kistler 1979) on others. It is not uncommon in the literature to find publications in which specific characteristics are misassigned to 1 subtype or the other. Therefore, here we use the Parseghian nomenclature (for obvious reasons) and make the necessary references to other nomenclatures to enlighten and not confuse (Parseghian et al. 1994; Parseghian and Hamkalo 2001).
One clear distinction that can be drawn between the core and linker histones is the relative immobility of the core histones (H2A, H2B, H3, and H4), compared with their linker counterparts (H1 and H5). Since 1974, it has become plain that linker histones are the nomads of the nucleus. The Ringertz laboratory, in work that has had far-reaching implications for the role of linker histones, created heterokaryons fusing HeLa cells with inactive chick erythrocyte nuclei. By radiolabelling HeLa proteins in a series of pulse-chase experiments, the researchers demonstrated that the decondensation and reactivation of a chick erythrocyte nucleus into a functional organelle occurred concomitant with the migration of human HeLa proteins into the chick nucleus (Appels et al. 1974a, 1974b). Among the histones, H1 was prominent in its ability to relocate to the chick nucleus and actually replace the avian linker histones already present there. The inhibition of DNA transcription and replication in the HeLas helped to verify that the presence of human histones in the chick nucleus was due to migration rather than to de novo synthesis in the cytoplasm. Furthermore, they were able to show that the synthesis of avian-specific genes was linked to the presence of human histones in the chicken counterpart. While this may sound contradictory to the oft held view that H1 plays a repressive role in transcription, one must view the various linker histones as having different degrees of repression. Replacing the chick erythrocyte H5 with a less repressive set of H1 subtypes can lead to a more open chromatin conformation. Perhaps nowhere is this better illustrated than in the work of Lu et al. (1999), who used the fully differentiated erythrocyte chromatin from quiescent nuclei in Xenopus. By incubating the erythrocyte chromatin in Xenopus egg extract, the researchers demonstrated the removal of endogenous H5 from the chromatin and replacement with an oocyte-specific H1 from the egg extract, facilitating the reacquisition of replicational competence (Lu et al. 1999). These observations extend the work of Dimitrov (Dimitrov and Wolffe 1996), who also demonstrated that replacement of somatic H1s and H1[degrees] with the oocyte H1 on Xenopus erythrocyte chromatin led to the re-acquisition of transcriptional competence.
Thus, histones, particularly linker histones, are not as sedentary as we once believed. In the case of linker histones, this was documented in observations using cells transfected with stably expressing histone--green fluorescent protein (GFP) fusions. These histone-GFP fusions would localize to the nucleus and fluoresce, but with the aid of a microscope, small areas of the nucleus could be photobleached. The time elapsed would then be measured until the recovery of fluorescence in the affected area by the repopulation of unbleached fusion molecules. This process, known as fluorescence recovery after photobleaching (FRAP) established the mobility of an H1 subtype, H1a (Doenecke's H1.1), and confirmed the importance of the carboxy terminal tail (Ctail) region of the protein in stabilizing its binding to DNA (Lever et al. 2000). It also established that phosphorylation is required for this mobility, although the target of phosphorylation may not be the H1. However, the need to phosphorylate an H1 for it to become mobile was quickly confirmed (Contreras et al. 2003). In a companion paper to Lever et al. (2000), researchers were able to determine a residence time for other H1 subtypes ([H1.sup.S]-1 and H1[degrees]) of about 220 s (~4 min) for each occupation of linker DNA (Misteli et al. 2000), but this value was called into question recently by the observation that GFP fusions to the C-tail render the H1 molecule less stable in vivo than fusions occurring on the amino terminal tail (N-tail; Th'ng et al. 2005). Relatively speaking, linker histones are far more sedentary than transcription factors. For instance, the mean residence times for the transcription factors HMGB1, Fos, and Jun range from about 1 to 30 s (Bianchi 2004; Phair et al. 2004). However, an equally important observation from the Misteli et al. (2000) paper was that 2 distinct pools of H1 subtypes exist, of which 1 is less mobile and associates with heterochromatin. This same observation was later confirmed independently (Contreras et al. 2003). While core histones are far more sedentary than linker histones, they too appear to have distinct pools of mobility (Kimura and Cook 2001; Kimura 2005). For instance, FRAP analysis reveals that a distinct minority of H2B-GFPs will exchange locations slowly ([t.sub.1/2] ~ 130 min), while the majority show no discernible mobility ([t.sub.1/2] > 8.5 h). Thus, some histone variants are apparently more nomadic than others. These observation harken to a model of H1 subtype distribution first proposed by Parseghian and Hamkalo (2001), in which heterochromatin is characterized by the presence of specific H1 subtypes that are absent from euchromatin. Support for such a model comes from studies in differential subtype distribution (Parseghian et al. 2000, 2001), differential gene expression (Alami et al. 2003), and differential subtype mobility (Th'ng et al. 2005).
In this model, the authors also propose that a particular subtype, [H1.sup.S]-1 (Doenecke's H1.2), is distributed throughout the nucleus and lays the foundation for a basal level of chromatin compaction. [H1.sup.S]-1 has several unique characteristics relevant to our discussion, including the highest turnover rate for any H1 and, unlike other histones, its expression is not restricted to S-phase (Pehrson and Cole 1982; Higurashi et al. 1987; Dominguez et al. 1992). Also, [H1.sup.S]-1 is enriched in soluble chromatin fractions (Huang and Cole 1984). Recent studies found [H1.sup.S]-1 to be less effective in aggregating nucleosomal arrays (Talasz et al. 1998), and identified it as 1 of the subtypes exhibiting a weaker binding affinity to DNA than that of most other H1s (Th'ng et al. 2005). Thus, it was not so surprising to us when researchers discovered a new role for this subtype as an apoptotic signaling agent when cells sustain double stranded DNA breaks (Konishi et al. 2003).
Beyond the walls of the nucleus
The presence of histones in the cytoplasm is not a newly discovered phenomenon, despite the recent observation that [H1.sup.S]-1 plays a central role in the induction of apoptosis by migrating from the nucleus to mitochondria. Perhaps the most often referenced work on extranuclear histones is that on the biochemical and immunofluorescence techniques employed by Zlatanova et al. (1990) to isolate cytoplasmic H1 in mouse liver and Friend erythroleukaemic cells. But a lesser known work published a year earlier uses a rather unique agent, an epidermolytic toxin conjugated to ferritin, to localize pools of H1 and H3 in the cytoplasm of 2 differentiated cells (Smith et al. 1989). The ferritin is bound to iron, which acts as an electron-dense signal for electron microscopy. While the researchers demonstrated the interaction of epidermolytic toxin to both H1 and H3 by Western analysis, the ferritin conjugate localizes their presence in the cytoplasmic regions of the stratum granulosum in skin and the stratum corneum in the eye. Given that these highly differentiated cells undergo nuclear dispersion, it may not be surprising that these histones are present in the cytoplasm. In recent years, our laboratory has confirmed these observations in rat and human tissue using anti-H1 monoclonal antibodies (M.H. Parseghian, unpublished data).
However, with the apoptosis studies of Konishi et al. …
Комментариев нет:
Отправить комментарий