Transcription of intergenic DNA deposits nucleosomes on promoter to silence gene expression

Abstract

Comment on: Hainer SJ, et al. Genes Dev. 2011; 25:29-40

Like all DNA-based processes, eukaryotic transcription takes place in the context of chromatin. The wrapping of 147 base pairs of DNA around an octamer of histone proteins (two copies each of H2A, H2B, H3 and H4) to form nucleosomes, the repeating unit of chromatin, can have dramatic effects on both transcription initiation and elongation. At promoters, nucleosomes interfere with the binding of sequence-specific transcription factors that may activate or repress their target genes.¹ Within the body of a gene, nucleosomes not only have a negative impact on transcription by physically impeding the progression of RNA Polymerase II (RNA Pol II), but also have a positive role in preventing aberrant initiation from cryptic promoters.¹ Not surprisingly, eukaryotic cells have co-opted mechanisms used to alter chromatin dynamics to control gene expression. One mechanism utilizes the activity of chromatin remodeling complexes, such as Swi/Snf and Isw2, to alter histone-DNA contacts in an ATP-dependent manner, ultimately leading to changes in nucleosome position or occupancy.^1,2 A second mechanism involves factors that modify histone residues through the addition or removal of acetyl, phosphoryl, methyl or ubiquityl groups. These histone modifications can alter nucleosome dynamics by either affecting histone-DNA interactions or influencing the recruitment and/or activity of additional transcription factors.^1,3 A third mechanism uses proteins known as histone chaperones, which facilitate the deposition and/or eviction of nucleosomes at both promoter and transcribed regions of the genome.^1,4 We have recently described a novel mechanism to control promoter chromatin dynamics that relies on transcription-coupled nucleosome assembly by histone chaperones.⁵

The Saccharomyces cerevisiae SER3 gene encodes an enzyme required for serine biosynthesis and its expression is tightly regulated by the availability of serine. Previous work discovered a noncoding RNA (ncRNA) whose transcription initiates within intergenic DNA 5′ of SER3 and extends across the SER3 promoter (Fig. 1), encompassing a region now referred to as SRG1 (SER3 Regulatory Gene 1).⁶ Results from several experiments indicate that transcription of SRG1, not the ncRNA product, represses SER3 by a transcriptional interference mechanism.⁶ Additional studies revealed that transcription of SRG1 mediates the serine-

Figure 1. Model for the repression of SER3 by SRG1 intergenic transcription. In the absence of serine, the Cha4 activator is bound to the SRG1 promoter but is unable to initiate transcription. The SER3 promoter is depleted of nucleosomes allowing proteins, either an as yet unknown sequence-specific activator or general transcription factors, to bind and activate SER3 transcription. In response to serine, Cha4 recruits SAGA and Swi/Snf to reposition the nucleosomes at the 5′ end of SRG1 toward the SER3 promoter, permitting initiation of SRG1 transcription. These repositioned nucleosomes are then disassembled ahead of the transcribing RNA Pol II and reassembled after passage of RNA Pol II by the Spt6 and Spt16 histone chaperones. The nucleosomes being maintained by SRG1 transcription occlude the SER3 promoter, preventing the binding of transcription factors and SER3 transcription.

dependent control of SER3 expression.⁷ In the presence of serine, the sequence-specific activator Cha4 recruits the Swi/Snf chromatin remodeler and the SAGA histone acetyltransferase to the SRG1 promoter. Together, these factors induce transcription of SRG1 across the SER3 promoter, inhibiting the binding of transcription factors required for SER3 expression. In the absence of serine, Cha4 no longer recruits SAGA and Swi/Snf, resulting in reduced SRG1 transcription that then allows transcription factors to bind and activate SER3 (Fig. 1).⁷

In a recent study, we investigated the transcription interference mechanism operating at SER3.⁵ We discovered that SRG1 transcription dramatically alters nucleosome dynamics at the SER3 promoter. Under conditions in which SRG1 is expressed and SER3 is repressed, nucleosomes are loosely positioned across the entire SRG1 transcription unit, including the SER3 promoter. When SRG1 transcription is reduced, SER3 activation is accompanied by a redistribution of nucleosomes, leaving the SER3 promoter free of nucleosomes.⁵ These results reveal a positive correlation between SRG1 transcription and nucleosome occupancy that contrasts with what has been generally observed for other transcribed yeast genes.¹ We then determined that mutations in SPT6 and SPT16, two genes encoding histone chaperones that facilitate nucleosome disassembly ahead of transcribing RNA Pol II and reassembly in its wake,^8-10 strongly derepress SER3 without disturbing SRG1 transcription. Moreover, we provided evidence that SER3 derepression in these mutants is accompanied by nucleosome depletion specifically over the SRG1 transcription unit and increased transcription factor binding to the SER3 promoter.⁵ Collectively, our data show that SRG1 transcription mediates SER3 repression, not simply because of the passage of RNA Pol II, but rather by assembling nucleosomes over the SER3 promoter (Fig. 1).

At first glance, SER3 repression by SRG1 transcription resembles a previously described mechanism whereby transcription-coupled nucleosome assembly by Spt6 and Spt16 prevents aberrant transcription initiation from cryptic promoters within the protein coding regions of many yeast genes.^10,11 Several studies have shown that repression of cryptic intragenic transcription also relies on a cascade of histone modifications, including Set2-dependent methylation of histone H3 lysine 36 and subsequent deacetylation of the N-terminal tail of histone H4.¹² Conversely, we have provided evidence that SER3 repression is completely independent of these histone modifications, suggesting these two mechanisms are distinct.⁵ At least two factors may contribute to the difference between these two mechanisms: the rate of transcription and the affinity of the transcribed DNA for histones. First, many of the genes that display cryptic initiation are lowly transcribed and thus may be more reliant on histone modifications to prevent aberrant intragenic transcription until the next RNA Pol II molecule arrives. In contrast, SRG1 is highly transcribed and the frequent passage of RNA Pol II may render these histone modifications irrelevant. Second, in contrast to most protein coding regions, promoter DNA is often rich in sequences that are refractory to nucleosomes.¹³ Therefore, the instability of nucleosomes assembled at the SER3 promoter as a result of SRG1 transcription may circumvent a possible role for histone modifications.

In recent years, extensive efforts by many laboratories have indicated that transcription in eukaryotes is not limited to protein coding genes, but rather occurs across entire genomes.¹⁴ As we move forward in understanding the functional significance of this widespread transcriptional activity, it is important to consider not only the astounding array of ncRNA products that it produces, but also its effect on chromatin architecture. The impact that transcription of ncDNA has on chromatin is likely to be relevant, not only to transcriptional regulation, but also to other cellular processes that are dependent on protein-DNA interactions.

Hainer SJ, Pruneski JA, Mitchell RD, Monteverde RM, Martens JA. Intergenic transcription causes repression by directing nucleosome assembly. Genes Dev. 2011;25:29–40. doi: 10.1101/gad.1975011.