In 2000, we proposed what has commonly come to be referred to as the ‘histone code hypothesis’, which, in its original form, posits that “multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions”.15
Parallels to François Jacob’s quote from “Evolution and Tinkering” are readily apparent. The same fixed set of amino acids that make up the histone proteins have the potential of being post-translationally modified within the chromatin template, where distinct spatiotemporal patterns of modifications ultimately shape functional outcome. One of the more striking phenomena predicted by such a code is that subtle variations to the same template can result in vastly different outcomes, especially in the context of regulation of gene expression.
At the time that we proposed the histone code hypothesis, we had a limited understanding of the true breadth of the number and type of PTMs that exist on histone residues either on the unstructured N-terminal tails that protrude from the nucleosomal surface or within the structured globular domains. Acetylation and phosphorylation were the best-characterized modifications at that time, with multiple sites and several of the enzymes responsible for their placement and removal having been identified. However, investigations on the dynamics of histone methylation were in their infancy. Only a handful of sites modified by methylation were known at the time, and the function of histone methylation was largely unclear, primarily because the enzyme systems responsible for the steady-state balance of methyl marks (histone methyltransferases and demethylases) were not yet identified and the intricacies associated with a modification that could exist in multiple states (mono-, di-, or trimethyl) complicated studies. Insight into other modifications was even more rudimentary. Today, we know that a number of PTMs exist, including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP-ribosylation, proline isomerization, citrullination, butyrylation, propionylation, and glycosylation ().11; 16; 17
Numerous studies using both biochemical and genetic approaches have revealed many of the enzymes that are responsible for placement or removal of these modifications on specific amino acid residues on histones as well as non-histone proteins. While the functional significance of some of these modifications remains to be determined, the collective field of chromatin biologists has made great strides toward identifying the biological consequence of others. For example, modifications can disturb contacts between histones in contiguous nucleosomes or histones with DNA, resulting in alteration of higher-order chromatin structure. Specifically, acetylation of lysine residues on histone tails neutralizes the basic charge of the residue on which it occurs, thereby disrupting histone contacts with other histones and/or DNA and in turn chromatin compaction.9
While it had been known that histone modifications such as methylation did not disrupt nucleosomal contacts by altering the charge of the modified residue, we now know that specialized domains within effector proteins facilitate recognition and binding to methyl marks in a defined state on specific residues to mediate downstream effects. Domains characterized thus far as being able to bind to methylated residues include chromodomains, tudor domains, PHD fingers, MBT domains, Ankyrin repeats, PWWP domains, HEAT domains and WD40 repeats ().18; 19; 20; 21; 22
Other domains that recognize and bind to specifically modified histone forms have also been characterized. For instance, where bromodomains can bind to acetylated lysine residues, 14-3-3, BRCT, and BIR domains can bind to phosphorylated threonine and serine residues ().19; 23
Histone modification types and the interacting domains that “read” them
The chromatin-modifying enzymes that facilitate alterations to the chromatin landscape by placing, removing, or interpreting modifications to establish variable states have been more recently come to be generally referred to as writers, erasers, and readers, respectively, of the histone code (). Returning to the idea of tinkering with chromatin, we are now in a position to appreciate the true potential of a “toolkit”24
of writers, erasers, and readers of the histone code in the establishment of proper spatiotemporal patterns of modifications necessary for cellular identity and function. At defined points, writers place marks on defined histone residues, which are in turn interpreted by readers harboring specialized domains that facilitate recognition and binding to the specific mark of interest to drive the progression of a specific biological phenomenon. At a time when such signaling needs to be terminated, erasers are recruited to their defined target(s) to remove the mark, thereby ending the associated functional outcome of the previously defined reader. Admittedly, the situation is made vastly more complicated by the fact that particular amino acid residues can house more than one type of modification (this is largely true for lysine residues, which can be methylated, acetylated, ubiquitylated, or sumoylated), and that some enzymes can write, erase, or read more than one modification. Moreover, one mark can often recruit multiple effector proteins.25; 26
Such complications, however, support the general notion of tinkering with combinatorial pattern of PTMs to control proper recruitment of effector proteins or complexes in which they reside.
Toolkit for modifying the chromatin template
We appreciate that the ‘histone code hypothesis’, as originally articulated by us in 2000, evolved into an influential review on the function(s) of covalent histone modifications. We acknowledge that this hypothesis, and extensions of it, rest heavily on the foundation of many biologists and biochemists who were dedicated to the general view that chromatin was going to be much more than a passive way to package the genome. However, because of the rapid pace of research in chromatin biology and the complexity associated with chromatin modifications such as those mentioned above, we must continually refine how we define the histone code. In fact, the mere existence of a code in the first place has been a point of contention.27
Beyond discussions in the field as to whether a strict histone code truly exists, there is also debate over whether it is most appropriate to define it as “code” in which definite combinations lead to an absolute outcome (as exemplified by the genetic code). Some see it more in terms of a “language”, where complex combinatorial patterns of modifications form words that ultimately give rise to a vocabulary of histone crosstalk.28
Others yet prefer to think of it more specifically in terms of an “epigenetic code” that is defined by combinations of histone PTMs which are predictive of, and necessary for, expression patterns of differentiation and developmental-specific genes.29
On the other hand, it has been argued that histone modifications are not truly “epigenetic”, as the nature of their heritability (a requisite condition to be defined in the classical sense of epigenetic) is questionable30
, thereby disputing the appropriateness of an “epigenetic code”. At some point, the question of how exactly to define the histone code becomes somewhat rhetorical, as at their very essence, all definitions ultimately seem to convey the same fundamental principle that histone PTMs act in concert to elicit downstream biological outcomes. Here we reflect on the many forms the ‘histone code hypothesis’ has come to take since the time of its inception a decade ago, and suggest that individual definitions may not be mutually exclusive of one another, but are perhaps instead complementary.