Biomedical subjects
Publications and source records attributed to Aseem Z Ansari.
As cellular machines and processes that regulate the flow of genomic information have come into sharper focus, a new level of chemical control has become possible. The scope of such chemical intervention extends from the mechanistic dissection of biochemical processes in living cells to the targeted control of gene networks and cell fate.
Functional engagement of RNA polymerase II (Pol II) with eukaryotic chromosomes is a fundamental and highly regulated biological process. Here we present a high-resolution map of Pol II occupancy across the entire yeast genome. We compared a wild-type strain with a strain bearing a substitution in the Sen1 helicase, which is a Pol II termination factor for noncoding RNA genes. The wild-type pattern of Pol II distribution provides unexpected insights into the mechanisms by which genes are repressed or silenced. Remarkably, a single amino acid substitution that compromises Sen1 function causes profound changes in Pol II distribution over both noncoding and protein-coding genes, establishing an important function of Sen1 in the regulation of transcription. Given the strong similarity of the yeast and human Sen1 proteins, our results suggest that progressive neurological disorders caused by substitutions in the human Sen1 homolog Senataxin may be due to misregulation of transcription.
Significant efforts have been devoted to the development of artificial transcriptional activators for use as mechanistic tools, as therapeutic agents, and for biomanufacturing applications. One of the primary challenges has been the development of artificial activators that exhibit potency in cells comparable to that of endogenous activators; the vast majority function only moderately in the cellular context. Here we demonstrate that the superimposition of two distinct binding modes, a masking interaction and an interaction with the transcriptional machinery, has a profoundly positive effect on the cellular activity of artificial activators, with up to 600-fold enhancement observed. Incorporation of this feature into future generations of small molecule transcriptional activators should increase their nuclear uptake and facilitate their accessibility to their target proteins, thus significantly augmenting both their activity and utility.
Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.
We investigated the binding of E. coli RNA polymerase holoenzymes bearing sigma70, sigma(S), sigma32, or sigma54 to the ribosomal RNA operons (rrn) in vivo. At the rrn promoter, we observed "holoenzyme switching" from Esigma70 to Esigma(S) or Esigma32 in response to environmental cues. We also examined if sigma factors are retained by core polymerase during transcript elongation. At the rrn operons, sigma70 translocates briefly with the elongating polymerase and is released stochastically from the core polymerase with an estimated half-life of approximately 4-7 s. Similarly, at gadA and htpG, operons that are targeted by Esigma(S) and Esigma32, respectively, we find that sigma(S) and sigma32 also dissociate stochastically, albeit more rapidly than sigma70, from the elongating core polymerase. Up to approximately 70% of Esigma70 (the major vegetative holoenzyme) in rapidly growing cells is engaged in transcribing the rrn operons. Thus, our results suggest that at least approximately 70% of cellular holoenzymes release sigma70 during transcript elongation. Release of sigma factors during each round of transcription provides a simple mechanism for rapidly reprogramming polymerase with the relevant sigma factor and is consistent with the occurrence of a "sigma cycle" in vivo.
Despite their enormous potential as novel research tools and therapeutic agents, artificial transcription factors (ATFs) that up-regulate transcription robustly in vivo remain elusive. In investigating an ATF that does function exceptionally well in vivo, we uncovered an unexpected relationship between transcription function and a binding interaction between the activation domain and an adjacent region of the DNA binding domain. Disruption of this interaction leads to complete loss of function in vivo, even though the activation domain is still able to bind to its target in the transcriptional machinery. We propose that this interaction parallels those between natural activation domains and their regulatory proteins, concealing the activation domain from solvent and the cellular milieu until it binds to its transcriptional machinery target. Inclusion of this property in the future design of ATFs should enhance their efficacy in vivo.
Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a "switch"-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.
The yeast cyclin-dependent kinase Srb10 phosphorylates various transcriptional activators as they activate transcription, and acidic transcriptional activating domains found on several activators directly bind Srb10. Here we show that the interaction between Srb10 (with its associated cyclin Srb11) and each of several different activating regions, in vitro, leads to the phosphorylation of peptide sequences attached to but outside of the activating regions themselves. In some cases, residues within the activating regions are also phosphorylated. The results define a mechanism by which a kinase is recruited to alternate substrates with diverse physiological consequences.
The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the beta' subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known sigma70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.
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Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD). The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors. Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro. In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together. Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II. Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex. Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified. In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation.
A polyamide-peptide conjugate is designed which recruits sequence specifically the developmental regulator Exd to a cognate DNA site. In particular, an eight-ring hairpin polyamide (Im-Im-Py(C3H6NHR)-Py-gamma-Im-Py-Py-Py-beta-Dp) with a heptapeptide (R = Ac-Phe-Tyr-Pro-Trp-Met-Lys-Gly-) attached on a central ring was shown to induce cooperative binding of the Drosophila Hox protein cofactor Exd with a Kd of 4.4 nM in vitro, an order of magnitude more efficient than the natural Hox protein partner Ubx. The conjugate joins two sequence specific domains, one for DNA and one for the protein. This small molecule thus serves as a cooperative protein-DNA dimerizer, which mimics the natural Hox family of developmental regulators.
We describe a set of RNA molecules that work as transcriptional activators when tethered to DNA. These RNA activating regions were found amongst a randomized set of molecules bearing variants of a 10 nt loop attached to an RNA stem. The various RNA activating regions all bear an identical five- residue sequence with an interspersed sixth residue. The result shows that although all natural activating regions characterized thus far are peptidic, this function can be served by other kinds of moieties as well.
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Typical eukaryotic transcriptional activators are composed of distinct functional domains, including a DNA binding domain and an activating domain. Artificial transcription factors have been designed wherein the DNA binding domain is a minor groove DNA binding hairpin polyamide linked by a flexible tether to short activating peptides, typically 16-20 residues in size. In this study, the linker between the polyamide and the peptide was altered in an incremental fashion using rigid oligoproline "molecular rulers" in the 18-45 A length range. We find that there is an optimal linker length which separates the DNA and the activation region for transcription activation.
Several yeast activators are phosphorylated by SRB10, a cyclin-dependent kinase associated with the transcriptional machinery. Sites of phosphorylation are found outside the activating region in each case, and the modification has different physiological consequences in different cases. We show here that certain acidic transcriptional activating regions contact SRB10 as assayed both in vivo and in vitro. The interaction evidently positions each activator, as it activates transcription, so that it gets phosphorylated by SRB10, and thus a common mechanism targets disparate substrates to the kinase.
P201 is a short (eight-residue) nonacidic peptide that comprises a strong transcriptional activating region when tethered to DNA in yeast. Here we identify the mediator protein Gal11 as an essential target of P201. Deletion of Gal11, which modestly decreases activation elicited by the typical acidic yeast activator, abolishes activation by DNA-tethered P201. A point mutation in Gal11, which has no effect on other Gal11 functions, also greatly diminishes activation by DNA-tethered P201. P201 binds to a fragment of Gal11 in vivo and in vitro, and the interaction is diminished by mutations in either component that decrease activation in vivo. P201, unlike the typical yeast acidic activating region, does not work in mammalian cells, which is consistent with the notion that the unique target of P201 (Gal11) is absent from mammalian cells.
Eukaryotic transcription factors are composed of interchangeable modules. This has led to the design of a wide variety of modular artificial transcription factors (ATFs) that can stimulate or inhibit the expression of targeted genes. The ability to regulate the expression of any targeted gene using a 'programmable' ATF offers a powerful tool for functional genomics and bears tremendous promise in developing the field of transcription-based therapeutics.