Nathan Gamarra

All Publications

• Integrated analysis of multimodal long-read epigenetic assays. bioRxiv : the preprint server for biology Marcus, J., Dixon-Luinenburg, O., Gamarra, N., Schwartz, J. P., Rozenwald, M., Maslan, A., Urnov, F. D., Straight, A. F., Altemose, N., Ioannidis, N. M., Streets, A. 2025

  Abstract

  Long-read sequencing assays that detect base modifications are becoming increasingly important research tools for the study of epigenetic regulation, especially with the development of DiMeLo-seq and similar methods that deposit non-native base modifications to mark a range of epigenetic features such as protein-DNA interactions and chromatin accessibility. A main benefit of these methods is their inherent capacity for multimodality, enabling the encoding of multiple genomic signals onto single nucleic acid molecules. However, there are limited tools available for visualization and statistical analysis of this type of multimodal data. Here we introduce dimelo-toolkit, a python package built to enable flexible visualizations and easy integration into custom data processing workflows. We demonstrate the utility of dimelo-toolkit's preset visualizations of multiple base modifications in long-read single-molecule sequencing data with a novel extension of the DiMeLo-seq protocol that can capture three separate aspects of chromatin state on the same single reads: target protein binding, CpG methylation, and chromatin accessibility. We apply this multimodal method to simultaneously map chromatin accessibility, CpG methylation, and LMNB1 and CTCF binding patterns, respectively, in GM12878 cells. Additionally, we use dimelo-toolkit to investigate technical biases that arise when working with this type of multimodal data. This software tool will pave the way for developing well-optimized protocols and help unlock previously inaccessible biological insights.

  View details for DOI 10.1101/2025.11.09.687458

  View details for PubMedID 41279073

  View details for PubMedCentralID PMC12637566

• DiMeLo-cito: a one-tube protocol for mapping protein-DNA interactions reveals CTCF bookmarking in mitosis. bioRxiv : the preprint server for biology Gamarra, N., Chittenden, C., Sundararajan, K., Schwartz, J. P., Lundqvist, S., Robles, D., Dixon-Luinenburg, O., Marcus, J., Maslan, A., Franklin, J. M., Streets, A., Straight, A. F., Altemose, N. 2025

  Abstract

  Genome regulation relies on complex and dynamic interactions between DNA and proteins. Recently, powerful methods have emerged that leverage third-generation sequencing to map protein-DNA interactions genome-wide. For example, Directed Methylation with Long-read sequencing (DiMeLo-seq) enables mapping of protein-DNA interactions along long, single chromatin fibers, including in highly repetitive genomic regions. However, DiMeLo-seq involves lossy centrifugation-based wash steps that limit its applicability to many sample types. To address this, we developed DiMeLo-cito, a single-tube, wash-free protocol that maximizes the yield and quality of genomic DNA obtained for long-read sequencing. This protocol enables the interrogation of genome-wide protein binding with as few as 100,000 cells and without the requirement of a nuclear envelope, enabling confident measurement of protein-DNA interactions during mitosis. Using this protocol, we detected strong binding of CTCF to mitotic chromosomes in diploid human cells, in contrast with earlier studies in karyotypically unstable cancer cell lines, suggesting that CTCF "bookmarks" specific sites critical for maintaining genome architecture across cell divisions. By expanding the capabilities of DiMeLo-seq to a broader range of sample types, DiMeLo-cito can provide new insights into genome regulation and organization.

  View details for DOI 10.1101/2025.03.11.642717

  View details for PubMedID 40161611

  View details for PubMedCentralID PMC11952428

• A hexasome is the preferred substrate for the INO80 chromatin remodeling complex, allowing versatility of function MOLECULAR CELL Hsieh, L. J., Gourdet, M. A., Moore, C. M., Munoz, E. N., Gamarra, N., Ramani, V., Narlikar, G. J. 2022; 82 (11): 2098-+

  Abstract

  The critical role of the INO80 chromatin remodeling complex in transcription is commonly attributed to its nucleosome sliding activity. Here, we have found that INO80 prefers to mobilize hexasomes over nucleosomes. INO80's preference for hexasomes reaches up to ∼60 fold when flanking DNA overhangs approach ∼18-bp linkers in yeast gene bodies. Correspondingly, deletion of INO80 significantly affects the positions of hexasome-sized particles within yeast genes in vivo. Our results raise the possibility that INO80 promotes nucleosome sliding by dislodging an H2A-H2B dimer, thereby making a nucleosome transiently resemble a hexasome. We propose that this mechanism allows INO80 to rapidly mobilize nucleosomes at promoters and hexasomes within gene bodies. Rapid repositioning of hexasomes that are generated in the wake of transcription may mitigate spurious transcription. More generally, such versatility may explain how INO80 regulates chromatin architecture during the diverse processes of transcription, replication, and repair.

  View details for DOI 10.1016/j.molcel.2022.04.026

  View details for Web of Science ID 000822389300003

  View details for PubMedID 35597239

  View details for PubMedCentralID PMC9351570

• Histone dynamics play a critical role in SNF2h-mediated nucleosome sliding NATURE STRUCTURAL & MOLECULAR BIOLOGY Gamarra, N., Narlikar, G. J. 2021; 28 (7): 548-+

  View details for DOI 10.1038/s41594-021-00620-7

  View details for Web of Science ID 000669766000001

  View details for PubMedID 34226739

  View details for PubMedCentralID PMC9040563

• Collaboration through chromatin: motors of transcription and chromatin structure JOURNAL OF MOLECULAR BIOLOGY Gamarra, N., Narlikar, G. J. 2021; 433 (14): 166876

  Abstract

  Packaging of the eukaryotic genome into chromatin places fundamental physical constraints on transcription. Clarifying how transcription operates within these constraints is essential to understand how eukaryotic gene expression programs are established and maintained. Here we review what is known about the mechanisms of transcription on chromatin templates. Current models indicate that transcription through chromatin is accomplished by the combination of an inherent nucleosome disrupting activity of RNA polymerase and the action of ATP-dependent chromatin remodeling motors. Collaboration between these two types of molecular motors is proposed to occur at all stages of transcription through diverse mechanisms. Further investigation of how these two motors combine their basic activities is essential to clarify the interdependent relationship between genome structure and transcription.

  View details for DOI 10.1016/j.jmb.2021.166876

  View details for Web of Science ID 000659159100003

  View details for PubMedID 33556407

  View details for PubMedCentralID PMC8989640

• Gene expression and cell identity controlled by anaphase-promoting complex NATURE Oh, E., Mark, K. G., Mocciaro, A., Watson, E. R., Prabu, J., Cha, D. D., Kampmann, M., Gamarra, N., Zhou, C. Y., Rape, M. 2020; 579 (7797): 136-+

  Abstract

  Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks1. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle2-5 but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters6,7, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division.

  View details for DOI 10.1038/s41586-020-2034-1

  View details for Web of Science ID 000517050000002

  View details for PubMedID 32076268

  View details for PubMedCentralID PMC7402266

• Organization of Chromatin by Intrinsic and Regulated Phase Separation CELL Gibson, B. A., Doolittle, L. K., Schneider, M. W. G., Jensen, L. E., Gamarra, N., Henry, L., Gerlich, D. W., Redding, S., Rosen, M. K. 2019; 179 (2): 470-+

  Abstract

  Eukaryotic chromatin is highly condensed but dynamically accessible to regulation and organized into subdomains. We demonstrate that reconstituted chromatin undergoes histone tail-driven liquid-liquid phase separation (LLPS) in physiologic salt and when microinjected into cell nuclei, producing dense and dynamic droplets. Linker histone H1 and internucleosome linker lengths shared across eukaryotes promote phase separation of chromatin, tune droplet properties, and coordinate to form condensates of consistent density in manners that parallel chromatin behavior in cells. Histone acetylation by p300 antagonizes chromatin phase separation, dissolving droplets in vitro and decreasing droplet formation in nuclei. In the presence of multi-bromodomain proteins, such as BRD4, highly acetylated chromatin forms a new phase-separated state with droplets of distinct physical properties, which can be immiscible with unmodified chromatin droplets, mimicking nuclear chromatin subdomains. Our data suggest a framework, based on intrinsic phase separation of the chromatin polymer, for understanding the organization and regulation of eukaryotic genomes.

  View details for DOI 10.1016/j.cell.2019.08.037

  View details for Web of Science ID 000488775900016

  View details for PubMedID 31543265

  View details for PubMedCentralID PMC6778041

• Cryo-EM structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome ELIFE Armache, J., Gamarra, N., Johnson, S. L., Leonard, J. D., Wu, S., Narlikar, G. J., Cheng, Y. 2019; 8

  Abstract

  The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn't show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation.

  View details for DOI 10.7554/eLife.46057

  View details for Web of Science ID 000474234700001

  View details for PubMedID 31210637

  View details for PubMedCentralID PMC6611695

• The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h ELIFE Gamarra, N., Johnson, S. L., Trnka, M. J., Burlingame, A. L., Narlikar, G. 2018; 7

  Abstract

  ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell.

  View details for DOI 10.7554/eLife.35322

  View details for Web of Science ID 000433415000001

  View details for PubMedID 29664398

  View details for PubMedCentralID PMC5976439

• Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Barajas, J. F., Finzel, K., Valentic, T. R., Shakya, G., Gamarra, N., Martinez, D., Meier, J. L., Vagstad, A. L., Newman, A. G., Townsend, C. A., Burkart, M. D., Tsai, S. 2016; 55 (42): 13005-13009

  Abstract

  In fungal non-reducing polyketide synthases (NR-PKS) the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism-based crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-of-function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.

  View details for DOI 10.1002/anie.201605401

  View details for Web of Science ID 000385799200007

  View details for PubMedID 27653519

  View details for PubMedCentralID PMC5161458

• Mechanisms of ATP-Dependent Chromatin Remodeling Motors ANNUAL REVIEW OF BIOPHYSICS, VOL 45 Zhou, C. Y., Johnson, S. L., Gamarra, N. I., Narlikar, G. J. edited by Dill, K. A. 2016; 45: 153-181

  Abstract

  Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.

  View details for DOI 10.1146/annurev-biophys-051013-022819

  View details for Web of Science ID 000382010600008

  View details for PubMedID 27391925

  View details for PubMedCentralID PMC9157391