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Second-Generation Method for Analysis of Chromatin Binding Using Formaldehyde Crosslinking Kinetics

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Second-Generation Method for Analysis of Chromatin Binding Using Formaldehyde Crosslinking Kinetics bioRxiv preprint doi: https://doi.org/10.1101/153353; this version posted June 21, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Second-generation crosslinking kinetic analysis Second-generation method for analysis of chromatin binding using formaldehyde crosslinking kinetics Hussain Zaidi1*, Elizabeth A. Hoffman2*, Savera J. Shetty2, Stefan Bekiranov2, and David T. Auble2 1From the School of Medicine Research Computing, University of Virginia, Charlottesville, VA 22908, USA 2From the Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA 22908, USA *Contributed equally Running title: Second-generation crosslinking kinetic analysis To whom correspondence should be addressed: David T. Auble, Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA 22908, USA. Telephone: (434) 243-2629; Fax: (434) 924-5069; Email: [email protected]; Stefan Bekiranov, Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA 22908, USA. Telephone: (434) 982-6631; Fax: (434) 924-5069; Email: [email protected] Keywords: chromatin immunoprecipitation (ChIP), protein dynamic, protein cross-linking, transcription factor, nucleic acid chemistry, chromatin structure, formaldehyde chemistry ABSTRACT and more robust glycine quench conditions. Formaldehyde crosslinking underpins Notably, we find that formaldehyde crosslinking many of the most commonly used experimental rates can vary dramatically for different protein- approaches in the chromatin field, especially in DNA interactions in vivo. Some interactions were capturing site-specific protein-DNA interactions. crosslinked much faster than the time scale for Extending such assays to assess the stability and macromolecular interaction, making them suitable binding kinetics of protein-DNA interactions is for kinetic analysis. For other interactions, we more challenging, requiring absolute find the crosslinking reaction occurred on the measurements with a relatively high degree of same time scale or slower than binding dynamics; physical precision. We previously described an for these it was in some cases possible to compute experimental framework called CLK, which uses the in vivo equilibrium-binding constant but not time-dependent formaldehyde crosslinking data to on- and off-rates for binding. Selected TATA- extract chromatin binding kinetic parameters. binding protein-promoter interactions displayed Many aspects of formaldehyde behavior in cells dynamic behavior on the minute to several are unknown or undocumented, however, and minutes time scale. could potentially impact analyses of CLK data. Here we report biochemical results that better define the properties of formaldehyde crosslinking Gene regulation is a complicated and in budding yeast cells. These results have the highly regulated process involving the coordinated potential to inform interpretations of ‘standard’ assembly of dozens of proteins on promoter DNA chromatin assays including chromatin within the context of chromatin (1–4). In vitro immunoprecipitation, and the chemical complexity studies have provided a structurally detailed we uncovered resulted in the development of an paradigm for how the transcription preinitiation improved method for measuring binding kinetics complex (PIC) is assembled and regulated (5–13), using the CLK approach. Optimum conditions but less is known about the dynamic assembly of included an increased formaldehyde concentration 1 bioRxiv preprint doi: https://doi.org/10.1101/153353; this version posted June 21, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Second-generation crosslinking kinetic analysis PICs in vivo or how transcription factors (TFs) obtained by different approaches. Support for the contribute kinetically to PIC assembly or to the CLK approach was obtained by measurement of rate of the initiation of synthesis of individual binding dynamics for two TFs with very different RNAs. To develop molecular models for how dynamic properties that had been assessed by live these processes occur in vivo, estimates of on- and cell imaging (15, 18, 19). However, live cell off-rates for TF binding to specific loci in vivo are imaging has its own technical challenges (20) and required. In instances in which kinetic in most cases it is not possible to identify measurements cannot be made, biophysically particular single copy chromatin sites of rigorous estimates of site-specific in vivo affinity interaction by live cell imaging (8, 21, 22). An (as opposed to estimates of relative affinity) and alternative approach is competition ChIP, an assay fractional occupancy would be valuable. that measures the rate of turnover between an Chromatin immunoprecipitation (ChIP) is endogenous and inducible copy of a TF. Our quite possibly the most widely used assay for recent work demonstrates that quantitative characterizing the interactions between TFs and estimates of locus-specific binding kinetics can be specific sites on chromatin. ChIP typically uses obtained by modeling competition ChIP data, formaldehyde to crosslink TFs to their chromatin including the estimation of residence times much sites (14), and while it is an undeniably powerful shorter than the time for full induction of the approach for determining transcription factor competitor TF (23). Importantly, comparison of binding locations with high precision (3), standard CLK and competition ChIP data for TATA- ChIP assays are static measurements that do not binding protein (TBP) to a few specific loci shows provide unambiguous insight into the in vivo that the time scales for chromatin interaction are kinetics of these dynamic interactions. Several similar as judged by the two methods, with assays have expanded ChIP to attempt to capture residence times for promoter binding being in these relationships. We previously developed a general on the order of several minutes (23). ChIP-based method, the crosslinking kinetics Nonetheless, locus-specific TF-chromatin (CLK) assay, which exploits the time dependence dynamics are just beginning to be explored, with of formaldehyde crosslinking to model chromatin- only a small number of TFs and chromatin sites TF binding dynamics on a broad time scale and at for which CLK, competition ChIP and/or live cell individual loci (15). In this approach, cells are imaging kinetic data are available. A key aspect incubated with formaldehyde for various periods of the CLK assay involves the trapping of bound of time, unreacted formaldehyde is then quenched, species using formaldehyde. Here we report and the extent of DNA site crosslinking of a TF of biochemical results that better define the chemical interest at each time point is quantified by ChIP. behavior of formaldehyde in yeast cells. An The time-dependent increase in ChIP signal results increased formaldehyde concentration led to more from a combination of time-dependent rapid crosslinking, which improved the time formaldehyde reactivity and time-dependent resolution and analytical ability of the assay to binding of free TF molecules to unoccupied DNA extract locus-specific binding kinetic information sites in the cell population. To distinguish kinetic for some TFs. For other TFs, an increased effects of crosslinking chemistry from kinetic formaldehyde concentration resulted in their effects of TF binding, measurements are made depletion from the soluble pool, and in some cases using congenic cells differing only in the rapid depletion. These observations emphasize the concentration of TF and the data are fit using both importance of optimizing the CLK approach for sets of data simultaneously (15, 16). analysis of the dynamic behavior of a particular A challenge with the development of TF. We report the development of a general and locus-specific kinetic assays such as CLK is that improved CLK method framework with both more aspects of the effects of formaldehyde on cells rapid crosslinking and more efficient quenching in largely remain a black box (17), and validation of yeast cells. We also report improved the extracted dynamic parameters is difficult computational methods for data analysis and because complementary approaches are still being describe improved approaches for distinguishing developed and there are few “gold standard” contributions of crosslinking rate and binding interactions with convergent kinetic measurements 2 bioRxiv preprint doi: https://doi.org/10.1101/153353; this version posted June 21, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Second-generation crosslinking kinetic analysis kinetics to the time-dependent increases in ChIP formaldehyde can be achieved with a higher signal. concentration of glycine. In addition to lower signals at each time RESULTS point obtained using max glycine conditions, some The CLK method relies on time-resolved time-dependent datasets showed initial shallow formaldehyde crosslinking
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