Raman Venkat Nelakanti

All Publications

• N6-methyladenine in DNA antagonizes SATB1 in early development. Nature Li, Z., Zhao, S., Nelakanti, R. V., Lin, K., Wu, T. P., Alderman, M. H., Guo, C., Wang, P., Zhang, M., Min, W., Jiang, Z., Wang, Y., Li, H., Xiao, A. Z. 2020; 583 (7817): 625-630

  Abstract

  The recent discovery of N6-methyladenine (N6-mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism1. However, the biological role of N6-mA and the molecular pathways that exert its function remain unclear. Here we show that N6-mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N6-mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD)2-4. Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures3,5,6. We show that the presence of N6-mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N6-mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N6-mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD-SATB1 interactions mediated by N6-mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N6-mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.

  View details for DOI 10.1038/s41586-020-2500-9

  View details for PubMedID 32669713

  View details for PubMedCentralID PMC8596487

• m(6)A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development IMMUNITY Gao, Y., Vasic, R., Song, Y., Teng, R., Liu, C., Gbyli, R., Biancon, G., Nelakanti, R., Lobben, K., Kudo, E., Liu, W., Ardasheva, A., Fu, X., Wang, X., Joshi, P., Lee, V., Dura, B., Viero, G., Iwasaki, A., Fan, R., Xiao, A., Flavell, R. A., Li, H., Tebaldi, T., Halene, S. 2020; 52 (6): 1007-+

  Abstract

  N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

  View details for DOI 10.1016/j.immuni.2020.05.003

  View details for Web of Science ID 000541890000015

  View details for PubMedID 32497523

  View details for PubMedCentralID PMC7408742

• Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA. Cell research Zhang, M., Yang, S., Nelakanti, R., Zhao, W., Liu, G., Li, Z., Liu, X., Wu, T., Xiao, A., Li, H. 2020; 30 (3): 197-210

  Abstract

  N6-methyladenine (N6-mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N6-mA undergo drastic fluctuation during early embryogenesis, indicative of active regulation. Here we show that the 2-oxoglutarate-dependent oxygenase ALKBH1 functions as a nuclear eraser of N6-mA in unpairing regions (e.g., SIDD, Stress-Induced DNA Double Helix Destabilization regions) of mammalian genomes. Enzymatic profiling studies revealed that ALKBH1 prefers bubbled or bulged DNAs as substrate, instead of single-stranded (ss-) or double-stranded (ds-) DNAs. Structural studies of ALKBH1 revealed an unexpected "stretch-out" conformation of its "Flip1" motif, a conserved element that usually bends over catalytic center to facilitate substrate base flipping in other DNA demethylases. Thus, lack of a bending "Flip1" explains the observed preference of ALKBH1 for unpairing substrates, in which the flipped N6-mA is primed for catalysis. Co-crystal structural studies of ALKBH1 bound to a 21-mer bulged DNA explained the need of both flanking duplexes and a flipped base for recognition and catalysis. Key elements (e.g., an ALKBH1-specific α1 helix) as well as residues contributing to structural integrity and catalytic activity were validated by structure-based mutagenesis studies. Furthermore, ssDNA-seq and DIP-seq analyses revealed significant co-occurrence of base unpairing regions with N6-mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N6-mA turnover of unpairing regions associated with dynamic chromosome regulation.

  View details for DOI 10.1038/s41422-019-0237-5

  View details for PubMedID 32051560

  View details for PubMedCentralID PMC7054317

• Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo. Cell stem cell Kooreman, N. G., Kim, Y. n., de Almeida, P. E., Termglinchan, V. n., Diecke, S. n., Shao, N. Y., Wei, T. T., Yi, H. n., Dey, D. n., Nelakanti, R. n., Brouwer, T. P., Paik, D. T., Sagiv-Barfi, I. n., Han, A. n., Quax, P. H., Hamming, J. F., Levy, R. n., Davis, M. M., Wu, J. C. 2018

  Abstract

  Cancer cells and embryonic tissues share a number of cellular and molecular properties, suggesting that induced pluripotent stem cells (iPSCs) may be harnessed to elicit anti-tumor responses in cancer vaccines. RNA sequencing revealed that human and murine iPSCs express tumor-associated antigens, and we show here a proof of principle for using irradiated iPSCs in autologous anti-tumor vaccines. In a prophylactic setting, iPSC vaccines prevent tumor growth in syngeneic murine breast cancer, mesothelioma, and melanoma models. As an adjuvant, the iPSC vaccine inhibited melanoma recurrence at the resection site and reduced metastatic tumor load, which was associated with fewer Th17 cells and increased CD11b+GR1himyeloid cells. Adoptive transfer of T cells isolated from vaccine-treated tumor-bearing mice inhibited tumor growth in unvaccinated recipients, indicating that the iPSC vaccine promotes an antigen-specific anti-tumor T cell response. Our data suggest an easy, generalizable strategy for multiple types of cancer that could prove highly valuable in clinical immunotherapy.

  View details for PubMedID 29456158

• ALKBH5 modulates hematopoietic stem and progenitor cell energy metabolism through m6A modification-mediated RNA stability control. Cell reports Gao, Y., Zimmer, J. T., Vasic, R., Liu, C., Gbyli, R., Zheng, S. J., Patel, A., Liu, W., Qi, Z., Li, Y., Nelakanti, R., Song, Y., Biancon, G., Xiao, A. Z., Slavoff, S., Kibbey, R. G., Flavell, R. A., Simon, M. D., Tebaldi, T., Li, H. B., Halene, S. 2023; 42 (10): 113163

  Abstract

  N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.

  View details for DOI 10.1016/j.celrep.2023.113163

  View details for PubMedID 37742191

  View details for PubMedCentralID PMC10636609

• ALKBH5 MODULATES HEMATOPOIETIC STEM AND PROGENITOR CELL ENERGY METABOLISM THROUGH M6A MODIFICATION -MEDIATED RNA STABILITY CONTROL Gao, Y., Biancon, G., Flavell, R., Gbyli, R., Halene, S., Kibbey, R., Li, H., Li, Y., Liu, C., Liu, W., Nelakanti, R., Patel, A., Qi, Z., Simon, M., Slavoff, S., Song, Y., Tebaldi, T., Vasic, R., Xiao, A., Zheng, S., Zimmer, J. ELSEVIER SCIENCE INC. 2023: S82

  View details for Web of Science ID 001057881700105

• A New Link to Primate Heart Development. Developmental cell Nelakanti, R. V., Xiao, A. Z. 2020; 54 (6): 685-686

  Abstract

  Long non-coding RNAs (lncRNAs) are important regulators of development. In this issue of Developmental Cell, Wilson et al. utilize pluripotent stem cell models to demonstrate that a primate lncRNA, BANCR, is primarily expressed in fetal cardiomyocytes and promotes cell migration.

  View details for DOI 10.1016/j.devcel.2020.09.009

  View details for PubMedID 32991832

• Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics. Cell reports Kooreman, N. G., de Almeida, P. E., Stack, J. P., Nelakanti, R. V., Diecke, S. n., Shao, N. Y., Swijnenburg, R. J., Sanchez-Freire, V. n., Matsa, E. n., Liu, C. n., Connolly, A. J., Hamming, J. F., Quax, P. H., Brehm, M. A., Greiner, D. L., Shultz, L. D., Wu, J. C. 2017; 20 (8): 1978–90

  Abstract

  There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

  View details for PubMedID 28834758

• Teratoma formation: a tool for monitoring pluripotency in stem cell research. Current protocols in stem cell biology Nelakanti, R. V., Kooreman, N. G., Wu, J. C. 2015; 32: 4A 8 1-4A 8 17

  Abstract

  This unit describes protocols for evaluating the pluripotency of embryonic and induced pluripotent stem cells using a teratoma formation assay. Cells are prepared for injection and transplanted into immunodeficient mice at the gastrocnemius muscle, a site well suited for teratoma growth and surgical access. Teratomas that form from the cell transplants are explanted, fixed in paraformaldehyde, and embedded in paraffin. These preserved samples are sectioned, stained, and analyzed. Pluripotency of a cell line is confirmed by whether the teratoma contains tissues derived from each of the embryonic germ layers: endoderm, mesoderm, and ectoderm. Alternatively, explanted and fixed teratomas can be cryopreserved for immunohistochemistry, which allows for more detailed identification of specific tissue types present in the samples. © 2015 by John Wiley & Sons, Inc.

  View details for DOI 10.1002/9780470151808.sc04a08s32

  View details for PubMedID 25640819