Zarmik Moqtaderi

Intrinsic histone-DNA interactions are not the major determinant of nucleosome positions in vivo.

We assess the role of intrinsic histone-DNA interactions by mapping nucleosomes assembled in vitro on genomic DNA. Nucleosomes strongly prefer yeast DNA over Escherichia coli DNA, indicating that the yeast genome evolved to favor nucleosome formation. Many yeast promoter and terminator regions intrinsically disfavor nucleosome formation, and nucleosomes assembled in vitro show strong rotational positioning. Nucleosome arrays generated by the ACF assembly factor have fewer nucleosome-free regions, reduced rotational positioning and less translational positioning than obtained by intrinsic histone-DNA interactions. Notably, nucleosomes assembled in vitro have only a limited preference for specific translational positions and do not show the pattern observed in vivo. Our results argue against a genomic code for nucleosome positioning, and they suggest that the nucleosomal pattern in coding regions arises primarily from statistical positioning from a barrier near the promoter that involves some aspect of transcriptional initiation by RNA polymerase II.

SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD+-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.

Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo

Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein‐protein and protein‐DNA cross‐links between molecules that are in close proximity on the chromatin template in vivo. DNA sequences that cross‐link with a given protein are selectively enriched, and reversal of the formaldehyde cross‐linking permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein‐protein and protein‐DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome‐wide basis. in this unit describes the ChIP procedure for Saccharomyces cerevisiae; describes the corresponding steps for mammalian cells.

Mapping accessible chromatin regions using Sono-Seq.

Disruptions in local chromatin structure often indicate features of biological interest such as regulatory regions. We find that sonication of cross-linked chromatin, when combined with a size-selection step and massively parallel short-read sequencing, can be used as a method (Sono-Seq) to map locations of high chromatin accessibility in promoter regions. Sono-Seq sites frequently correspond to actively transcribed promoter regions, as evidenced by their co-association with RNA Polymerase II ChIP regions, transcription start sites, histone H3 lysine 4 trimethylation (H3K4me3) marks, and CpG islands; signals over other sites, such as those bound by the CTCF insulator, are also observed. The pattern of breakage by Sono-Seq overlaps with, but is distinct from, that observed for FAIRE and DNase I hypersensitive sites. Our results demonstrate that Sono-Seq can be a useful and simple method by which to map many local alterations in chromatin structure. Furthermore, our results provide insights into the mapping of binding sites by using ChIP–Seq experiments and the value of reference samples that should be used in such experiments.

Close association of RNA polymerase II and many transcription factors with Pol III genes

Transcription of the eukaryotic genomes is carried out by three distinct RNA polymerases I, II, and III, whereby each polymerase is thought to independently transcribe a distinct set of genes. To investigate a possible relationship of RNA polymerases II and III, we mapped their in vivo binding sites throughout the human genome by using ChIP-Seq in two different cell lines, GM12878 and K562 cells. Pol III was found to bind near many known genes as well as several previously unidentified target genes. RNA-Seq studies indicate that a majority of the bound genes are expressed, although a subset are not suggestive of stalling by RNA polymerase III. Pol II was found to bind near many known Pol III genes, including tRNA, U6, HVG, hY, 7SK and previously unidentified Pol III target genes. Similarly, in vivo binding studies also reveal that a number of transcription factors normally associated with Pol II transcription, including c-Fos, c-Jun and c-Myc, also tightly associate with most Pol III-transcribed genes. Inhibition of Pol II activity using α-amanitin reduced expression of a number of Pol III genes (e.g., U6, hY, HVG), suggesting that Pol II plays an important role in regulating their transcription. These results indicate that, contrary to previous expectations, polymerases can often work with one another to globally coordinate gene expression.

Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells

Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III–associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

Genome-Wide Occupancy Profile of the RNA Polymerase III Machinery in Saccharomyces cerevisiae Reveals Loci with Incomplete Transcription Complexes

ABSTRACT We used chromatin immunoprecipitation, followed by microarray hybridization, to determine the genome-wide distribution of the RNA polymerase (Pol) III transcription apparatus in the yeast Saccharomyces cerevisiae. The Pol III transcriptome includes all tRNA genes, previously identified non-tRNA Pol III genes, and SNR52, which encodes a small nucleolar RNA. Unexpectedly, we identify eight ETC loci that are occupied by TFIIIC but not by other components of the Pol III machinery. Some ETC loci contain stretches of DNA that are highly conserved among closely related yeast species, suggesting that they may encode functional RNAs. ETC6 is located upstream of the gene encoding the τ 91 subunit of TFIIIC, suggesting the possibility of Pol III-regulated expression of a critical Pol III factor. We also identify the ZOD1 locus, which is bound by all components of the Pol III machinery and yet does not appear to express an RNA conserved among closely related yeast species. The B block motifs and several flanking nucleotides of the ZOD1 and ETC loci are very similar to each other and are highly conserved across the yeast species. Furthermore, the unusual profile of Pol III factor association with ZOD1 and the ETC loci is perfectly preserved in a different Saccharomyces species, indicating that these loci represent novel functional entities.

The TAFs in the HAT

The striking phenomenon of histone acetylases with histone-like substructures that play critical (though distinct) roles in eukaryotic gene regulation raises evolutionary questions. We propose the following speculative scenario that begins with an Archea-like organism containing TBP and histones lacking N-terminal tails. Following the split between Archea and eukaryotes, we imagine a eukaryotic ancestor with histone tails and a histone acetylase that might conceivably be associated with the histones; such an organism would have the ability to modify nucleosome structure. Subsequently, gene duplications and evolutionary divergence results in two sets of histones; the standard nucleosomal histones and the histone-like TAFs that associate with the histone acetylase while losing the ability to form nucleosomes. Next, the primitive histone acetylase complexes diverge into two types, which are distinguished by the catalytic subunit (Gcn5 or TAF-HAT). The TAF-HAT type acquired the ability to interact with TBP, additional TAF subunits, and promoter DNA, whereas the Gcn5 type acquired Ada and Spt subunits to facilitate the interaction with (and hence modification of) nucleosomal histones. Further divergence after the yeast–human split generated novel TAF-like proteins (e.g., PAF65α and PAF65β) that are specific to the human PCAF and hGcn5 complexes. In considering these ideas, it would be of interest to examine very primitive eukaryotes for the presence of histones, TAFs, Spt and Ada proteins, and Gcn5-like histone acetylases.

Activator-Specific Recruitment of TFIID and Regulation of Ribosomal Protein Genes in Yeast

In yeast, TFIID strongly associates with nearly all ribosomal protein (RP) promoters, but a TAF-independent form of TBP preferentially associates with other active promoters. RP promoters are regulated in response to growth stimuli, in most cases by a Rap1-containing activator. This Rap1-dependent activator is necessary and sufficient for TFIID recruitment, whereas other activators do not efficiently recruit TFIID. TAFs are recruited to RP promoters even when TBP and other general transcription factors are not associated, suggesting that TFIID recruitment involves a direct activator-TAF interaction. Most RP promoters lack canonical TATA elements, and they are preferentially activated by the Rap1-containing activator. These results demonstrate activator-specific recruitment of TFIID in vivo, and they suggest that TFIID recruitment is important for coordinate expression of RP genes.

The Histone H3–like TAF Is Broadly Required for Transcription in Yeast

In yeast cells, independent depletion of TAFs (130, 67, 40, and 19) found specifically in TFIID results in selective effects on transcription, including a common effect on his3 core promoter function. In contrast, depletion of TAF17, which is also present in the SAGA histone acetylase complex, causes a decrease in transcription of most genes. However, TAF17-depleted cells maintain Ace1-dependent activation, and they induce de novo activation by heat shock factor in a manner predominantly associated with the activator, not the core promoter. Thus, TAF17 is broadly, but not universally, required for transcription in yeast, TAF17 depletion and TAF130 depletion each disrupt TFIID integrity yet cause different transcriptional consequences, suggesting that the widespread influence of TAF17 might not be due solely to its function in TFIID.