Histone exchange is associated with activator function at transcribed promoters and with repression at histone loci

Transcription in eukaryotes correlates with major chromatin changes, including the replacement of old nucleosomal histones by new histones at the promoters of genes. The role of these histone exchange events in transcription remains unclear. In particular, the causal relationship between histone exchange and activator binding, preinitiation complex (PIC) assembly, and/or subsequent transcription remains unclear. Here, we provide evidence that histone exchange at gene promoters is not simply a consequence of PIC assembly or transcription but instead is mediated by activators. We further show that not all activators up-regulate gene expression by inducing histone turnover. Thus, histone exchange does not simply correlate with transcriptional activity, but instead reflects the mode of action of the activator. Last, we show that histone turnover is not only associated with activator function but also plays a role in transcriptional repression at the histone loci

Uncoupling histone turnover from transcription-associated histone H3 modifications

Transcription in eukaryotes is associated with two major changes in chromatin organization. Firstly, nucleosomal histones are continuously replaced by new histones, an event that in yeast occurs predominantly at transcriptionally active promoters. Secondly, histones become modified post-translationally at specific lysine residues. Some modifications, including histone H3 trimethylation at lysine 4 (H3K4me3) and acetylation at lysines 9 (H3K9ac) and 14 (H3K14ac), are specifically enriched at active promoters where histones exchange, suggesting a possible causal relationship. Other modifications accumulate within transcribed regions and one of them, H3K36me3, is thought to prevent histone exchange. Here we explored the relationship between these four H3 modifications and histone turnover at a few selected genes. Using lysine-to-arginine mutants and a histone exchange assay, we found that none of these modifications plays a major role in either promoting or preventing histone turnover. Unexpectedly, mutation of H3K56, whose acetylation occurs prior to chromatin incorporation, had an effect only when introduced into the nucleosomal histone. Furthermore, we used various genetic approaches to show that histone turnover can be experimentally altered with no major consequence on the H3 modifications tested. Together, these results suggest that transcription-associated histone turnover and H3 modification are two correlating but largely independent events

Hepatitis B Virus X Protein and Simian Virus 5 V Protein Exhibit Similar UV-DDB1 Binding Properties To Mediate Distinct Activities

The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2

The Oct-1 POU domain activates snRNA gene transcription by contacting a region in the SNAP(c) largest subunit that bears sequence similarities to the Oct-1 coactivator OBF-1

The RNA polymerases II and III snRNA gene promoters contain an octamer sequence as part of the enhancer and a proximal sequence element (PSE) as part of the core promoter. The octamer and the PSE bind the POU domain activator Oct-1 and the basal transcription factor SNAP(c), respectively. Oct-1, but not Oct-1 with a single E7R mutation within the POU domain, binds cooperatively with SNAP(c) and, in effect, recruits SNAP(c) to the PSE. Here, we show that SNAP(c) recruitment is mediated by an interaction between the Oct-1 POU domain and a small region of the largest subunit of SNAP(c), SNAP190. This SNAP190 region is strikingly similar to a region in the B-cell-specific Oct-1 coactivator, OBF-1, that is required for interaction with octamer-bound Oct-1 POU domain. The Oct-1 POU domain–SNAP190 interaction is a direct protein–protein contact as determined by the isolation of a switched specificity SNAP190 mutant that interacts with Oct-1 POU E7R but not with wild-type Oct-1 POU. We also show that this direct protein–protein contact results in activation of transcription. Thus, we have identified an activation target of a human activator, Oct-1, within its cognate basal transcription complex

Independent RNA polymerase II preinitiation complex dynamics and nucleosome turnover at promoter sites in vivo

Transcription by all three eukaryotic RNA polymerases involves the assembly of a large preinitiation complex (PIC) at gene promoters. The PIC comprises several general transcription factors (GTFs), including TBP, and the respective RNA polymerase. It has been suggested that some GTFs remain stably bound at active promoters to facilitate multiple transcription events. Here we used two complementary approaches to show that, in G1-arrested yeast cells, TBP exchanges very rapidly even at the most highly active RNA Pol II promoters. A similar situation is observed at RNA Pol III promoters. In contrast, TBP remains stably bound at RNA Pol I promoters. We also provide evidence that, unexpectedly, PIC dynamics are neither the cause nor the consequence of nucleosome exchange at most of the RNA Pol II promoters we analyzed. These results point to a stable reinitiation complex at RNA Pol I promoters and suggest independent PIC and nucleosome turnover at many RNA Pol II promoters

Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase

Nucleosomes are surprisingly dynamic structures in vivo, showing transcription-independent exchange of histones H2A-H2B genome-wide and exchange of H3-H4 mainly within the promoters of transcribed genes. In addition, nucleosomes are disrupted in front of and reassembled behind the elongating RNA polymerase. Here we show that inactivation of histone chaperone Spt16 in yeast results in rapid loss of H2B and H3 from transcribed genes but also from inactive genes. In all cases, histone loss is blocked by a transcription inhibitor, indicating a transcription-dependent event. Thus, nucleosomes are efficiently evicted by the polymerase but do not reform in the absence of Spt16. Yet exchange of nucleosomal H2B with free histones occurs normally, and, unexpectedly, incorporation of new H3 increases at all loci tested. This points to Spt16 restoring normal nucleosome structure by redepositing the displaced H3-H4 histones, thereby preventing incorporation of new histones and perhaps changes in histone modification patterns associated with ongoing transcription