Control of Chromatin Structure by Spt6: Different Consequences in Coding and Regulatory Regions

Abstract

Spt6 is a highly conserved factor required for normal transcription and chromatin structure. To gain new insights into the roles of Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant. We found that the level of nucleosomes is greatly reduced across some, but not all, coding regions in an spt6 mutant, with nucleosome loss preferentially occurring over highly transcribed genes. This result provides strong support for recent studies that have suggested that transcription at low levels does not displace nucleosomes, while transcription at high levels does, and adds the idea that Spt6 is required for restoration of nucleosomes at the highly transcribed genes. Unexpectedly, our studies have also suggested that the spt6 effects on nucleosome levels across coding regions do not cause the spt6 effects on mRNA levels, suggesting that the role of Spt6 across coding regions is separate from its role in transcriptional regulation. In the case of the CHA1 gene, regulation by Spt6 likely occurs by controlling the position of the ؉1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve an independent function.The regulation of transcription by chromatin dynamics is dependent upon distinct classes of factors that function by interacting with nucleosomes. One set of factors directly modifies histones, the protein components of nucleosomes, by either adding or removing covalent modifications, such as methyl or acetyl groups; these modifications can regulate transcription in a positive or negative fashion (12, 61). A second set of factors is comprised of chromatin-remodeling complexes, which control transcription by regulating the occupancy or positions of nucleosomes in an ATP-dependent fashion (16). A third set of factors are believed to act as histone chaperones. These factors function both in transcription initiation, serving to facilitate the removal or deposition of nucleosomes over regulatory regions, and in transcription elongation, to remove nucleosome barriers before transcribing RNA polymerase (RNAP) II or to redeposit nucleosomes in the wake of transcription (9,20). The integration of the activities of these different classes of factors is critical in transcriptional regulation. Spt6, a putative histone chaperone and a member of the third class, is the subject of this paper.Spt6 is a highly conserved protein that has been previously shown to control chromatin structure and transcription. Originally identified as an essential factor required for normal transcription in Saccharomyces cerevisiae (17, 54, 55), Spt6 has also been shown to play critical or essential roles in mammalian cells (73, 74), zebrafish (36, 39, 68), Drosophila (5, 23), nematodes (56), Schizosaccharomyces pombe (77), and Candida albicans (2). In addition to chromatin structure and transcription, Spt6 functions in recombination (51...

Chromatin and Transcription in Yeast

“…This has been observed globally for Asf1 Kaplan et al 2008), Rtt106 (Imbeault et al 2008), and the CAF-1 and Hir complexes (Rosa et al 2010), although spt16 mutants, as cited above, do show increased turnover (Jamai et al 2009). Similarly, in spt6 mutants there is slow histone redeposition at PHO5 , and nucleosomes are generally depleted from highly transcribed genes (Ivanovska et al 2011). It is important to note that steadystate turnover studies report on both eviction and replacement and thus do not distinguish between these two processes, but mutants that preferentially affect histone incorporation over eviction are expected to exhibit decreased nucleosome occupancy Ivanovska et al 2011), while the converse will be true of mutants that preferentially act in histone eviction.…”

“…Similarly, in spt6 mutants there is slow histone redeposition at PHO5 , and nucleosomes are generally depleted from highly transcribed genes (Ivanovska et al 2011). It is important to note that steadystate turnover studies report on both eviction and replacement and thus do not distinguish between these two processes, but mutants that preferentially affect histone incorporation over eviction are expected to exhibit decreased nucleosome occupancy Ivanovska et al 2011), while the converse will be true of mutants that preferentially act in histone eviction. Furthermore, in activation/repression paradigms (e.g., PHO5 induction) the two processes can be disentangled.…”

“…Genetic and biochemical studies have suggested that Spt6 controls transcription initiation, elongation, and 39 end formation. However, although Spt6 associates with coding regions genome-wide Ivanovska et al 2011), there is little understanding of what makes transcription of some genes Spt6 dependent and others not (Ivanovska et al 2011). With respect to initiation, spt6 mutants are defective for nucleosome reassembly or positioning over some promoter regions during transcriptional repression Jensen et al 2008;Ivanovska et al 2011), and spt6 mutations suppress some promoter insertions or deletions (Winston et al 1984;Prelich and Winston 1993).…”

“…The region of Spt6 required for interaction with nucleosomes is in the amino-terminal region and overlaps with the region required for Spt6-Spn1 interactions (McDonald et al 2010). In vivo, Spt6 is required to maintain a normal level of nucleosomes across highly transcribed coding regions (Kaplan et al 2003;Ivanovska et al 2011). The Spt6-chromatin connection is also supported by genetic interactions: spt6 mutations suppress the loss of the Swi/ Snf chromatin-remodeling complex (Neigeborn et al 1986(Neigeborn et al , 1987Bortvin and Winston 1996), and spt6 mutations themselves are suppressed by elevated levels of histone H3 (Bortvin and Winston 1996).…”

ASF1 and the SWI/SNF complex interact functionally during nucleosome displacement, while FACT is required for nucleosome reassembly at yeast heat shock gene promoters during sustained stress

“…The idea that in vitro nucleosome assembly/disassembly can be separated into several intermediate steps suggests that each of these steps can be aided by different histone chaperones (Elsasser and D'Arcy 2012). While FACT remains a primary candidate for the H2A-H2B dimer removal/deposition, the chaperoning of H3-H4 histones can be disputed between ASF1 (English et al 2005(English et al , 2006 and Spt6 (Ivanovska et al 2011) and is likely gene specific. The difficulty in discerning a specific mechanism for the individual chaperones can be partially explained by the presence of unstructured regions that are highly pliable and can participate in multiple target interactions utilizing an "induced fit" (Elsasser and D'Arcy 2012).…”