Silent chromatin (heterochromatin) is often associated with repetitive DNA sequences near centromeres or telomeres, and plays important roles in transcriptional regulation and chromosome segregation 
. Heterochromatin has been assumed to fold into a compacted structure 
, and the level of compaction can be modulated by histone modifications 
. The popular perception is that a compacted chromatin structure inhibits gene expression. However, recent studies using cryo-EM 
, ESI (electron spectroscopic imaging) 
, and 3C (chromosome conformation capture) 
suggest that the basic structure of active and silent chromatin during interphase is formed by extended 11 nm nucleosome arrays instead of compacted 30 nm fibers, as was previously suggested 
. Intriguingly, the incubation of purified SIR proteins with purified yeast chromatin is shown to promote the in vitro
formation of a heterochromatin structure based on extended 11 nm fibers 
. These observations imply that the formation of heterochromatin could occur without chromatin compaction. The precise structure of heterochromatin and the mechanism of gene silencing continue to remain elusive.
Studies in yeast, fly and mammals have suggested divergent mechanisms for the assembly of heterochromatin, but there are certain analogous features in the repressive mechanisms in these organisms 
. One common theme is that heterochromatin mediated gene silencing can spread along chromosomes 
. For example, HP1 is implicated in driving heterochromatin assembly in fly and mammals. HP1 is shown to bind to nucleosomes methylated at histone H3 K9. HP1 in turn recruits a histone methyltransferase, Suv39, that specifically methylates H3 K9 of adjacent nucleosomes. This promotes further HP1 binding, thereby leading to an iterative cycle that enables the spreading of heterochromatin 
. Telomeric heterochromatin in budding yeast propagates from a nucleation process via Rap1 binding at chromosome tips. Rap1 in turn recruits the silent information regulator (SIR) complex 
. The Sir2 subunit then deacetylates histones H3 and H4 of neighboring nucleosomes, promoting additional SIR complex binding 
. This initiates recurrent rounds of histone deacetylation and SIR binding, leading to the spreading of silenced chromatin.
The SIR complex is able to associate with specific nucleosomes within silent chromatin, but the molecular mechanism of how this association occurs is poorly understood. The binding sites of SIR are proposed to be formed by the highly conserved N-terminal tails and globular domains of H3 and H4 
. Deacetylation of H4 K16 in the H4-N terminus is particularly crucial for Sir3 binding in vivo
and in vitro 
. Besides acetylation, histone methylation is involved in regulating the spreading of silent chromatin in budding yeast. H3 K4 and K79 methylations catalyzed by Set1 or Dot1 respectively are thought to prevent promiscuous binding of SIR at loci other than the sub-telomeric regions 
In addition to H3 and H4 N-termini, the conserved H2B C-terminus also contributes to telomeric silencing 
. The crystal structure of the yeast nucleosome core particle predicts that inter-nucleosomal contacts are made by the H2B αC helix (hereafter simplified as H2B αC) because this extremely well ordered H2B αC is crucial in defining the surface of the nucleosome 
. The sole modification identified at H2B αC is the monoubiquitylation of lysine 123, located at the highly conserved AVTKY motif 
. As such, the dynamic regulation of H2B K123 ubiquitylation (H2Bub1) serves as a good candidate to shape chromatin structure, by modulating inter-nucleosomal interactions 
. However, it is not known whether the H2B αC has a bona fide
function in regulating SIR binding and higher-order organization of silent chromatin.
Here, we have investigated the role of the H2B αC in the assembly of heterochromatin in vivo, through the use of yeast strains that carry mutations in the residues of H2B αC. Our experiments using genetic analysis, bacterial dam methylase access and sucrose gradient sedimentation, all indicate a unique role of H2B αC in silent chromatin assembly, independent of H2Bub1. Surprisingly, we find that telomeric chromatin is assembled into a nucleosomal array with a regular alignment that requires H2B T122. The replacement of H2B T122 with glutamic acid induces disorderly chromatin compaction specifically at the telomere, and invasion of euchromatic histone marks. The results suggest that the organization of telomeric chromatin may be based on an extended chromatin fiber in vivo.