About

Jonathan Henninger, Ph.D., is an Assistant Professor and Principal Investigator in the Department of Biological Sciences at Carnegie Mellon University, where he joined in January 2024. He earned his B.S. in Biochemistry from the Indiana University of Pennsylvania before pursuing his Ph.D. in Developmental and Regenerative Biology at Harvard University. During his doctoral studies, he was mentored by Dr. Leonard Zon and focused on studying cellular mechanisms of blood development and blood cancer using zebrafish as a model system. Following his Ph.D., Dr. Henninger conducted postdoctoral research at the Whitehead Institute for Biomedical Research at MIT in the laboratory of Dr. Richard Young. His postdoctoral work involved discovering how RNA directly regulates its own transcription. Dr. Henninger's research integrates developmental biology and molecular mechanisms to understand gene regulation and blood cell development.

Research topics

• Biology
• Genetics
• Computer Science
• Physics
• Medicine
• Internal medicine
• Computational biology
• Cell biology
• Biophysics

Selected publications

• APE1 condensation in nucleoli of non-cancer cells depends on rRNA transcription and forming G-quadruplex RNA structures

  Nucleic Acids Research · 2025-02-27 · 8 citations

  articleOpen access

  APE1 [apurinic/apyrimidinic (AP) endodeoxyribonuclease 1] is the main endonuclease of the base excision repair pathway acting on abasic (AP) sites in DNA. APE1 is an abundant nuclear protein, and improper expression or localization of this factor could lead to the accumulation of toxic DNA intermediates. Altered APE1 subcellular distribution and expression are associated with cancer development, suggesting the importance of a fine-tuning mechanism for APE1 activities. Recent works highlighted the presence of APE1 within nucleoli of cancer cells and the ability of APE1 to form biomolecular condensate. However, whether secondary structures of ribosomal RNA (rRNA) influence the nucleolar localization of APE1 remains poorly understood. Since protein overexpression can result in artificial nucleolar accumulation, it is imperative to have appropriate cellular models to study APE1 trafficking under physiological conditions. To address this issue, we generated a murine embryonic stem cell line expressing endogenous fluorescent-tagged APE1. Live-cell imaging demonstrates that APE1 nucleolar accumulation requires active rRNA transcription and is modulated by different genotoxicants. In vitro experiments showed that APE1 condensate formation depends on RNA-forming G-quadruplex structures and relies on critical lysine residues. This study sheds light on the mechanisms underlying APE1 trafficking to the nucleolus and the formation of RNA-dependent APE1 nucleolar condensates.

  PublisherDOI

• Transient gene melting governs the timing of oligodendrocyte maturation

  Cell · 2025-08-25 · 2 citations

  articleOpen access
  PublisherOA PDFDOI

• CRISPR-Cas13d functional transcriptomics reveals widespread isoform-selective cancer dependencies on lncRNAs

  Blood · 2025-05-22 · 4 citations

  articleOpen access

  ABSTRACT: Long noncoding RNAs (lncRNAs) are a significant yet largely uncharted component of the cancer transcriptome, with their isoform-specific functions remaining poorly understood. In this study, we used RNA-targeting CRISPR-Cas13d to uncover and characterize hundreds of tumor-essential lncRNA (te-lncRNA) isoforms with clinical relevance. Focusing on multiple myeloma (MM), we targeted the lncRNA transcriptome expressed in tumor cells from patients with MM and revealed both MM-specific and pan-cancer dependencies across diverse cancer cell lines, which we further validated in animal models. Additionally, we mapped the subcellular localization of these te-lncRNAs, identifying >30 cytosolic isoforms that proved essential when targeted by cytosol-localized Cas13d. Notably, a specific isoform of small nucleolar RNA host gene 6, enriched in the endoplasmic reticulum, interacts with heat shock proteins to maintain cellular proteostasis. We also integrated functional and clinical data into the publicly accessible LongDEP Portal, providing a valuable resource for the research community. Our study offers a comprehensive characterization of te-lncRNAs, underscoring their oncogenic roles and therapeutic potential.

  PublisherOA PDFDOI

• An RNA-centric view of transcription and genome organization

  Molecular Cell · 2024-10-01 · 44 citations

  reviewOpen access1st authorCorresponding
  PublisherOA PDFDOI

• Active RNA synthesis patterns nuclear condensates

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-13 · 14 citations

  preprintOpen access

  Biomolecular condensates are membraneless compartments that organize biochemical processes in cells. In contrast to well-understood mechanisms describing how condensates form and dissolve, the principles underlying condensate patterning - including their size, number and spacing in the cell - remain largely unknown. We hypothesized that RNA, a key regulator of condensate formation and dissolution, influences condensate patterning. Using nucleolar fibrillar centers (FCs) as a model condensate, we found that inhibiting ribosomal RNA synthesis significantly alters the patterning of FCs. Physical theory and experimental observations support a model whereby active RNA synthesis generates a non-equilibrium state that arrests condensate coarsening and thus contributes to condensate patterning. Altering FC condensate patterning by expression of the FC component TCOF1 impairs ribosomal RNA processing, linking condensate patterning to biological function. These results reveal how non-equilibrium states driven by active chemical processes regulate condensate patterning, which is important for cellular biochemistry and function.

  PublisherOA PDFDOI

• Nucleolar accumulation of APE1 through condensates is mediated by rRNA forming G-quadruplex structures

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-03-05

  preprintOpen access

  Abstract APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is the main endonuclease of the base excision repair (BER) pathway acting on abasic (AP)-sites in damaged DNA. APE1 is an abundant nuclear protein with a higher concentration than other BER pathway enzymes, and therefore, improper expression and localization of this factor could lead to the accumulation of toxic DNA intermediates. Altered APE1 sub-cellular localization, expression levels, or hyper-acetylation are associated with cancer development suggesting the importance of a fine-tuning mechanism for APE1 nuclear-associated processes. Recent work highlighted multi-functional roles of APE1, including rRNA quality control. However, how rRNA influences the sub-cellular localization and activity of APE1 remains poorly understood, but previously underappreciated APE1-RNA interactions may influence the ability of this protein to form biomolecular condensates and tune APE1 partitioning into nucleoli. Since nucleolar accumulation of ectopic proteins could be the result of overexpression strategies, it is imperative to have cellular models to study APE1 trafficking under physiological conditions. Here we created the first cell line to express fluorescently tagged APE1 at its endogenous locus, enabling live-cell imaging. Live-cell imaging demonstrates that APE1 nucleolar accumulation requires active rRNA transcription. When modeled in vitro, APE1 condensate formation depends on RNA G-quadruplex (rG4) structures in rRNA and is modulated by critical lysine residues of APE1. This study sheds light on the mechanisms underlying APE1 trafficking to the nucleolus and formation of RNA-dependent APE1 nucleolar condensates that may modulate a switch between the activity of this factor in rRNA processing and DNA damage repair. Significance Statement We created and characterized the first endogenous, fluorescently tagged cell line to study APE1 subcellular trafficking under physiological and stress conditions. Using this cell line, we show that APE1 nucleolar enrichment occurs under physiological conditions and, performing in vitro droplet assays, we associate APE1 condensates with active transcription of RNA G-quadruplexes, abundantly present in healthy nucleoli. This work deepens our understanding of APE1’s role in healthy cells in the absence of DNA damage and provide a novel mechanism for how this protein responds to stress. Our results suggest that phase separation is an important part of how DNA damage repair proteins switch between their normal physiological functions and their ability to correct DNA lesions.

  PublisherOA PDFDOI

• Transcription factors interact with RNA to regulate genes

  Molecular Cell · 2023-07-01 · 255 citations

  articleOpen access
  PublisherOA PDFDOI

• Bioprocessing of MIR17HG Results in Long and Short Noncoding RNAs with Targetable Tumor-Promoting Activity in Multiple Myeloma

  Blood · 2022-11-15 · 1 citations

  article

  Our recent studies demonstrated that multiple myeloma (MM) cells can become significantly addicted to and in turn therapeutically susceptible to the modulation of oncogenic noncoding RNAs (ncRNAs). Through large-scale CRISPR interference (CRISPRi) and CRISPR-Cas13d viability screens, we identified MIR17HG as a leading cell growth-dependency in 6 MM cell lines. MIR17HG is mainly known for producing the miRNA cluster miR-17-92, which includes six mature oncogenic miRNAs (-17, -18a, -19a, -20a, -19b, -92a). However, we observed that depleting MIR17HG pre-RNA with sgRNAs or antisense oligonucleotides (ASO) took >3 days to downregulate the miR-17-92 members, yet appreciable transcriptional changes were immediately produced followed by cell cycle arrest in G0-G1 in 10 MM cell lines and CD138+ cells from 13 MM patients. Moreover, antagonism of miR-17-92 using anti-miRs did not phenocopy the anti-MM activity of targeting the MIR17HG pre-RNA, and this activity was only partially rescued by ectopic expression of miR-17-92 mimics. Therefore, we reasoned that alternative transcripts generated at this locus could provide cell growth dependency. We analyzed our RNA-seq dataset of CD138+ cells from >300 MM patients and 70 MM cell lines for these transcripts and observed reads from regions of MIR17HG that are not included in miR-17-92. Using qRT-PCR and single molecule RNA FISH, we identified the expression of a nuclear-enriched, ~5k-nt long, polyA(+) lncRNA. This lncRNA, named lnc-17-92 was quickly downregulated within a few hours of exposure to ASOs targeting the MIR17HG pre-RNA. Using DROSHA-knockout MM cells, in which MIR17HG produces lnc-17-92 but not miR-17-92, we found that targeting the MIR17HG pre-RNA effectively antagonized MM cell growth. Using easy-to-manipulate cellular models (e.g., HCT-116), we observed stronger rescue by ectopic expression of lnc-17-92, as compared to miR-17-92, after targeting MIR17HG pre-RNA. Using RNA-protein pull down (RPPD) and RNA yeast-3-hybrid (Y3H) assays, we found that the transcription factor c-MYC and the epigenetic modulator WDR82 were binding partners for lnc-17-92. In fact, we found that lnc-17-92 mediated their protein-protein interaction by providing the chromatin scaffold for the assembly of a MYC-WDR82 complex at the promoter region of ACC1. This gene encodes the limiting enzyme for de novo lipogenesis, a metabolic pathway that we show is controlled by lnc-17-92 to promote MM cell growth. Using three different cellular models (P493-6, HMECMYC, and U226MYC), we found that targeting MIR17HG pre-RNA preferentially killed MYC+ cells. This could be explained both by lnc-17-92 acting as a chromatin scaffold for MYC and also the known role of miR-17-92 in fine-tuning the downstream transcriptional program of MYC. However, we also observed that lnc-17-92 protected MYC from proteasomal degradation and found that the miR-17-92 member miR-92a negatively regulated MYC protein expression via an, as yet, unknown mechanism. The concerted action of lnc-17-92 and miR-17-92 on MYC protein stability is under investigation and updates will also be presented. Finally, we explored MIR17HG as a therapeutic target, which includes targeting both its lncRNA and miRNAs. To develop clinically applicable inhibitors, we screened >80 fully phosphorothioated (PS), 2'-O-methoxyethyl (2'-MOE)-modified, lipid-conjugated ASOs that could either trigger RNase H-mediated degradation of MIR17HG pre-RNA (gapmeRs) or exert function via an RNase H-independent mechanism (blockmeRs). We identified an 18-mer tocopherol (T)-conjugated gapmeR G2-15b-T ("G") and an 18-mer tocopherol (T)-conjugated steric blocker SB9-19-T ("B"). We found they had potent anti-tumor effects both in vitro and in vivo in three pre-clinical animal models, including a clinically relevant PDX-NSG mouse model. Tumor growth inhibition ranged from 100% (regression) to 50% in these models. Inhibitors did not cause overt toxicity in the mice, as shown by blood cell count, clinical, and body weight analysis. These inhibitors are currently being developed for translation to clinical trials. Altogether, our studies characterize a novel oncogenic mechanism for MIR17HG and provide clinically applicable inhibitors.

  PublisherDOI

• A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth

  Blood · 2022-09-20 · 35 citations

  articleOpen access

  Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

  PublisherOA PDFDOI

• Differential cofactor dependencies define distinct types of human enhancers

  Nature · 2022-06-01 · 109 citations

  articleOpen access
  PublisherOA PDFDOI

Recent grants

• Identifying factors that promote clonal dominance in zebrafish hematopoiesis

  NIH · $98k · 2015–2017

Frequent coauthors

• Leonard I. Zon

  Boston Children's Museum

  83 shared

• Elliott J. Hagedorn

  Boston University

  50 shared

• Brian Li

  Cleveland Clinic

  50 shared

• Raquel Riquelme

  Harvard University

  50 shared

• Richard A. Young

  34 shared

• Jessica L. Moore

  Yale University

  33 shared

• Deborah T. Hung

  Broad Institute

  30 shared

• Christoph M. Ernst

  German Center for Infection Research

  30 shared

Labs

• Henninger LabPI

Education

• Ph.D., Developmental and Regenerative Biology

  Harvard University

• Other, MIT

  Whitehead Institute for Biomedical Research