The HAT complex SAS-I has previously been described to acetylate H4 K16 in subtelomeric regions, and that this counteracts the spreading of telomeric heterochromatin (11
). This study substantially refines and expands our view by providing a high-resolution map of global H4 K16Ac by SAS-I. First, our data show that SAS-I acetylation is not specifically targeted to subtelomeric sequences, suggesting that the increase in SIR spreading in sas2Δ
is the result of increased affinity of the SIR complex to hypoacetylated chromatin rather than to the absence of a specific, local boundary. Secondly, our data reveal the surprising finding that in the absence of Sas2, there is a depletion of H4 K16Ac in the majority of ORFs of the yeast genome, whereas intergenic regions show little dependence on Sas2. Thus, in sas2Δ
cells, most H4 K16Ac that is deposited by other HATs remains in the promoter and 5′ region of genes. The regions most susceptible to Sas2 acetylation also show low transcription and low histone exchange rates. Thus, a picture emerges where Sas2 acetylation of H4 K16Ac is deposited at a given time/in a given process in the genome, and that this pattern is subsequently ‘sculptured’ by transcription- independent histone exchange in promoter regions and by passage of the transcription machinery per se
within ORFs. Thus, SAS-I shows a different mode of operation than other histone-modifying complexes in that it is not recruited to promoters, nor does it migrate along ORFs in association with PolII. Since SAS-I interacts with the chromatin assembly factor CAF-I (9
), which performs DNA replication- coupled chromatin assembly, we propose that SAS-I performs genome-wide H4 K16 acetylation during S-phase, in the wake of chromatin assembly. After replication, if transcription and histone exchange rates are low, H4 K16Ac by SAS-I remains in chromatin after deposition. Conversely, if exchange and transcription are high, this will lead to a decrease in H4 K16Ac, because the histones incorporated outside of S-phase and replication-coupled chromatin assembly are underacetylated on H4 K16. One consequence of this model is that Sas2 might only transiently be associated with chromatin, which is in agreement with the fact that we (data not shown) and others (17
) have not been able to find Sas2 associated at telomeres of ORFs by ChIP analysis, although this may be due to technical limitations.
Asf1 is an H3/H4 chaperone that mediates transcription-dependent histone exchange (5
). Interestingly, SAS-I also interacts with Asf1 (8
), suggesting that SAS-I might perform histone acetylation coupled to Asf1-dependent histone deposition. Future experiments will be required to test this and assess the functional relevance of this interaction.
Since loss of H4 K16Ac in ORFs is so pervasive in sas2Δ
cells, does this have a global effect on transcription? The most prominent effect of sas2Δ
is reduced expression of subtelomeric genes due to SIR spreading [(11
), data not shown]. Notably, we also found sas2Δ
to be resistant to 6-AU, which is indicative of an effect on transcription elongation (25
). However, this did not translate easily to a biochemically measurable variable. We found a mild increase in PolII processivity, but no change in PolII elongation rate, and the level of transcript from the 3′-end of genes was slightly higher in sas2Δ
. However, this effect seemed to be distinct from the initiation of cryptic transcripts from within genes, which leads to the generation of shorter transcripts at selected genes that have a defined start site and a defined length (30
). Also, the increase of 3′ transcript levels in sas2Δ
was milder than that observed in other cryptic initiation mutants like set2Δ
, or in the absence of the HDAC complex Rpd3(S) (32
), and unlike set2Δ
or the deletion of Rpd3 complex components, sas2Δ
exacerbated the cryptic initiation defect of mutations in SPT6
at a FLO8::HIS3
reporter construct, but had no effect on its own. In sum, these results suggested distinct mechanisms of altering transcription between SAS-I and other factors affecting cryptic initiation, and the exact nature of this alteration by SAS-I is unclear.
We furthermore observed a more pronounced loss of H4 K16Ac at long than at short genes in sas2Δ
. One possibility is that acetylation in the 5′ region of short genes is performed by different HAT(s) than that of long genes, and that this/these HATs have a broader range of acetylation. Alternatively, there may be differences in transcription-coupled acetylation on long versus short genes that become evident in the absence of Sas2. Which HATs contribute to the remainder of H4 K16Ac in sas2Δ
is unclear. There is a partial contribution of Esa1/NuA4 to H4 K16Ac at telomeres (12
), but bulk H4 K16Ac is not decreased in esa1
). Thus, a systematic analysis of H4 K16Ac in sas2Δ
mutants combined with deletions in other HATs will be required to identify the enzymes responsible for residual H4 K16Ac in sas2Δ
What purpose does genome-wide Sas2-dependent H4 K16 acetylation serve? Perhaps this is a way to mark the chromatin as duplicated after DNA replication and chromatin assembly, thus maintaining it in a euchromatic state. Since H4 K16Ac prevents SIR binding (13
), instituting this mark in S-phase might prevent inappropriate SIR binding in other regions of the genome particularly during the process of chromatin assembly, when histone modifications otherwise are diluted.
In summary, this study reveals a novel global role for SAS-I-mediated histone acetylation in ORFs throughout the genome and relates it to histone exchange and transcription elongation. H4 K16Ac has previously been linked to elongation in other organisms. However, in mammalian cells, phosphorylation of H3 Ser10 during transcriptional activation leads to the recruitment of MOF, which is a close homologue of Sas2 and a HAT for H4 K16. This in turn modulates transcription elongation via recruitment of BRD4 (35
). Furthermore, MOF in Drosophila
directly binds in a bimodal fashion to the promoters and 3′-end of dosage-compensated genes on the male X-chromosome and increases their expression (36
). Thus, MOF in these organisms is targeted to its site of action using a different mechanism than SAS-I in yeast. Therefore, although these enzymes are structurally homologous and show the same substrate specificity, there are important species-specific mechanistic differences in their function and effect on chromatin.