All Publications

  • Production of Homogeneous, Functional Zinc-Finger Arrays in High Yield With Two Chromatographic Steps. Bio-protocol Liang, J., Azubel, M., Wang, G., Nie, Y., Kornberg, R. D., Beel, A. J., Matteï, P. J. 2025; 15 (16): e5420

    Abstract

    Zinc-finger (ZF) arrays are compact, sequence-specific polynucleotide-binding domains, which have been used to target the delivery of diverse effector domains, enabling applications such as gene identification, localization, regulation, and editing. To facilitate in vitro applications of ZF arrays, we have developed a general method for their expression and purification. Here, we describe a protocol involving two chromatographic steps that yields homogeneous and functional ZF arrays in milligram quantities. Key features • A general method for expressing and purifying C2H2 ZF arrays in E. coli, compatible with both natural and artificial ZFs. • The His-SUMO tag improves ZF-array solubility, eliminating the need for denaturation and refolding steps. • Simple, two-step purification yields milligram-scale ZF arrays suitable for downstream applications.

    View details for DOI 10.21769/BioProtoc.5420

    View details for PubMedID 40873471

    View details for PubMedCentralID PMC12378415

  • A universal method for the purification of C2H2 zinc finger arrays. PloS one Liang, J., Azubel, M., Wang, G., Nie, Y., Kornberg, R. D., Beel, A. J., Mattei, P. J. 2025; 20 (2): e0318295

    Abstract

    Zinc fingers (ZFs) are compact, modular, sequence-specific polynucleotide-binding domains uniquely suited for use as DNA probes and for the targeted delivery of effector domains for purposes such as gene regulation and editing. Despite recent advances in both the design and application of ZF-containing proteins, there is still a lack of a general method for their expression and purification. Here we describe a simple method, involving two chromatographic steps, for the production of homogeneous, functional ZF proteins in high yield (one milligram per liter of bacterial culture), and we demonstrate the generality of this method by applying it to a diverse set of eight C2H2-type ZF proteins. By incorporating a surface-exposed terminal cysteine residue that enables site-specific conjugation with maleimide-activated fluorophores, we confirm the suitability of these probes for in situ labeling of specific DNA sequences in human cells.

    View details for DOI 10.1371/journal.pone.0318295

    View details for PubMedID 39903729

    View details for PubMedCentralID PMC11793764

  • Structure of mitotic chromosomes. Molecular cell Beel, A. J., Azubel, M., Mattei, P., Kornberg, R. D. 2021

    Abstract

    Chromatin fibers must fold or coil in the process of chromosome condensation. Patterns of coiling have been demonstrated for reconstituted chromatin, but the actual trajectories of fibers in condensed states of chromosomes could not be visualized because of the high density of the material. We have exploited partial decondensation of mitotic chromosomes to reveal their internal structure at sub-nucleosomal resolution by cryo-electron tomography, without the use of stains, fixatives, milling, or sectioning. DNA gyres around nucleosomes were visible, allowing the nucleosomes to be identified and their orientations to be determined. Linker DNA regions were traced, revealing the trajectories of the chromatin fibers. The trajectories were irregular, with almost no evidence of coiling and no short- or long-range order of the chromosomal material. The 146-bp core particle, long known as a product of nuclease digestion, is identified as the native state of the nucleosome, with no regular spacing along the chromatin fibers.

    View details for DOI 10.1016/j.molcel.2021.08.020

    View details for PubMedID 34520722

  • 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science (New York, N.Y.) Hoencamp, C., Dudchenko, O., Elbatsh, A. M., Brahmachari, S., Raaijmakers, J. A., van Schaik, T., Sedeño Cacciatore, Á., Contessoto, V. G., van Heesbeen, R. G., van den Broek, B., Mhaskar, A. N., Teunissen, H., St Hilaire, B. G., Weisz, D., Omer, A. D., Pham, M., Colaric, Z., Yang, Z., Rao, S. S., Mitra, N., Lui, C., Yao, W., Khan, R., Moroz, L. L., Kohn, A., St Leger, J., Mena, A., Holcroft, K., Gambetta, M. C., Lim, F., Farley, E., Stein, N., Haddad, A., Chauss, D., Mutlu, A. S., Wang, M. C., Young, N. D., Hildebrandt, E., Cheng, H. H., Knight, C. J., Burnham, T. L., Hovel, K. A., Beel, A. J., Mattei, P. J., Kornberg, R. D., Warren, W. C., Cary, G., Gómez-Skarmeta, J. L., Hinman, V., Lindblad-Toh, K., Di Palma, F., Maeshima, K., Multani, A. S., Pathak, S., Nel-Themaat, L., Behringer, R. R., Kaur, P., Medema, R. H., van Steensel, B., de Wit, E., Onuchic, J. N., Di Pierro, M., Lieberman Aiden, E., Rowland, B. D. 2021; 372 (6545): 984-989

    Abstract

    We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

    View details for DOI 10.1126/science.abe2218

    View details for PubMedID 34045355

  • Role of mediator in transcription control Robinson, P., Trnka, M. J., Pellarin, R., Bushnell, D. A., Greenberg, C., Davis, R. E., Mattei, P., Sali, A., Burlingame, A. L., Kornberg, R. D. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2017: S41

    View details for Web of Science ID 000407623600058

  • Chromatin potentiates transcription. Proceedings of the National Academy of Sciences of the United States of America Nagai, S., Davis, R. E., Mattei, P. J., Eagen, K. P., Kornberg, R. D. 2017; 114 (7): 1536-1541

    Abstract

    Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.

    View details for DOI 10.1073/pnas.1620312114

    View details for PubMedID 28137832

    View details for PubMedCentralID PMC5320956

  • Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex. Cell Robinson, P. J., Trnka, M. J., Bushnell, D. A., Davis, R. E., Mattei, P., Burlingame, A. L., Kornberg, R. D. 2016; 166 (6): 1411-1422 e16

    Abstract

    A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

    View details for DOI 10.1016/j.cell.2016.08.050

    View details for PubMedID 27610567

  • Uncoupling Promoter Opening from Start-Site Scanning. Molecular cell Murakami, K., Mattei, P., Davis, R. E., Jin, H., Kaplan, C. D., Kornberg, R. D. 2015; 59 (1): 133-138

    Abstract

    Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.

    View details for DOI 10.1016/j.molcel.2015.05.021

    View details for PubMedID 26073544

    View details for PubMedCentralID PMC4490988