Research

Learn more about the major research areas in the Rao laboratory:
 

  1. Regulation of NFAT and NFkB, key transcription factors in activated T cells
  2. Identification of critical players in store-operated calcium entry
  3. Alternate transcriptional programs activated by NFAT and their relevance to cancer immunotherapy
  4. Regulation of gene expression in T and B cells
  5. Discovery of the TET family of DNA methylcytosine oxidases

 

Our lab has worked on several fundamental pathways of gene expression in T cells and other cell types (e.g. B cells, embryonic stem cells). Two current areas that are directly relevant to cancer are: A, mechanisms of cancer initiation and progression, and B, cancer immunotherapy (see Figure).

A. In the cancer arena, we are investigating the role of TET (Ten-Eleven Translocation) enzymes and their partner proteins in a process known as heterochromatin dysfunction, that appears to be a prelude to a sequence of cellular events that operates in premalignancy and cancer as well as cellular senescence and aging (see below). We have also just begun to investigate the roles of these enzymes and certain partner proteins in postmitotic neurons.

B. In the cancer immunotherapy arena, we are investigating the transcriptional networks in tumour-infiltrating immune cells (T cells, T regulatory cells, myeloid cells) that modulate the balance between an active versus an attenuated immune response to the tumour. The ultimate objective is to develop strategies to counter T cell exhaustion, dial down immunosuppression by T regulatory cells and myeloid cells, and promote the immune destruction of cancers.

In both areas, our experiments utilize mouse models as well as an extensive suite of whole-genome analyses that require bioinformatic analyses. These include RNA-sequencing, including for nascent RNA; whole-genome / whole-exome sequencing; chromatin immunoprecipitation for transcription factors and histone modifications; chromatin accessibility mapping by ATAC-sequencing; chromosome conformation capture (Hi-C); whole-genome bisuphite sequencing; and base-resolution and DNA pulldown methods to map 5hmC and other oxidised methylcytosines generated by TET enzymes. We are interested in identifying and characterizing proteins that modulate TET activity both physically and functionally, through mass spectrometry and whole-genome screens. We are also involved in characterizing these complexes structurally through cryo-EM, X-ray crystallography and NMR as appropriate.
 

 

 

A. Mechanisms of Cancer Initiation and Progression

1. The TET (Ten-Eleven Translocation) family of DNA methylcytosine oxidases

In collaboration with Dr. L. Aravind (NCBI), we showed that proteins of the TET family are dioxygenases that utilize Fe(II), molecular oxygen, and a-ketoglutarate (also known as 2-oxoglutarate) to oxidize the methyl group of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) [1, 2] and other oxidised methylcytosines (oxi-mC) in DNA (see Figure). This discovery identified TET enzymes as the long-sought effectors of DNA demethylation, and spurred the development of new methods for genome-wide mapping of 5hmC and other oxi-mC in DNA [3, 4]. We have examined the roles of TET proteins and 5hmC in the immune and haematopoietic systems and during embryonic development and oncogenesis. TET enzymes are now established as essential players in all known pathways of DNA cytosine demethylation, and as critical regulators of gene expression, cell lineage specification, embryonic development, neuronal function and cancer [5].
 

     

  1. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by the MLL fusion partner, TET1. Science 2009; 324: 930-935. [pdf] – pls link to published version on our site, merged main text and suppl
  2. Ko M*, Huang Y*, Jankowska AM, Pape UJ, Tahiliani M, Bandukwala HS, Ahn J, Lamperti ED, Koh KP, Ganetzky R, Liu XS, Aravind L, Agarwal S, Maciejewski J, Rao A. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 2010; 468: 839-843. [pdf]
  3. Pastor WA, Aravind L, Rao A. TETonic shift: Biological roles of TET proteins in DNA demethylation and transcription. Nature Reviews Mol Cell Biol 2013: 14: 341-356. PMC3804139 [pdf]
  4. Pastor WA*, Pape UJ*, Huang Y*, Henderson HR, Lister R, Ko MG, McLoughlin EM, Brudno Y, Maha-patra S, Kapranov P, Tahiliani M, Daley GQ, Liu XS, Ecker JR, Milos PM, Agarwal S, Rao A. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 2011, 473: 394-397. [pdf]
  5. Lio C-W J*, Yue X, López-Moyado IF, Tahiliani M, Aravind L, Rao A. TET methylcytosine oxidases: new insights from a decade of research. J Biosci 2020; 45: 21. [pdf]