Page 67 - Biennial Report 2018-20 Jun 2021

This study suggests that chronic systemic IL6 can indeed be a cause for fatty liver and that this
may be achieved through dysregulation of glycolysis/gluconeogenesis pathway. This may be the
driving force behind the obesity-independent fatty liver observed in many Indians.

DELINEATING AND MODULATING THE FUNCTION OF Ep300 IN THE MOLECULAR
PATHOLOGY OF RUBINSTEIN TAYBI SYNDROME

The objectives of this study by Chetana Sachidanandan included; first, to create a mutant
zebrafish line for Ep300; second, to define the molecular pathophysiology of the RSTS zebrafish
model; third, to probe the regulatory relationship between progenitor genes and Ep300; fourth,
to look for neural crest defects in the RSTS model and fifth, to identify small molecules that can
compensate for Ep300 loss in the RSTS model.
Two regions in EP300a and Ep300b were identified to create mutations based on their high
degree of conservation and functional importance. Guide RNAs were designed for each of these
regions to create CRISPR-Cas mutants. The guide RNA designed against the HAT domain of
Ep300a protein was injected in 1-cell stage embryos. These embryos were then grown for a few
days to test whether any mutations are being generated. This was done by heteroduplex assays.
After establishing that mutations were being generated, the HD guide RNA was injected embryos
were grown to adulthood. These animals were further fin-clipped and assayed for genomic
changes in the locus. Genomic changes leading to differential migration in heteroduplex assays
were found. The process of amplifying and sequencing the DNA from putative mutant fish is
being done.
A number of heteroduplex positive mutants in their heterozygous state were genotyped and
sequenced and it was realized that the heteroduplex assay does not correlate well with
mutations in the F2 generation. The genotyping and sequencing were expanded to the larger
number of F1 fishes to identify multiple independent mutations. Meanwhile zebrafish embryos,
injected with antisense morpholinos against Ep300a, are also being characterized for further
molecular phenotyping in specialized tissues such as the neural crest.
It was discovered that the putative mutants that were identified based on heteroduplex assays
did not show genomic changes. It is suspected that the strong mutant phenotype might have
caused early lethality. A fresh batch of fishes with injection of various concentrations of single
and double guide RNAs to create selective mutations in the HAT domain and the Bromodomain
are now being created. Meanwhile, zebrafish embryos injected with antisense morpholinos
against Ep300a were characterized and an early defect in neural crest migration was discovered.
Further markers are now being analyzed to delineate the molecular phenotypes resulting from
neural crest migration problems.

TARGETING TELOMERASE TO TACKLE AGGRESSIVE DRUG RESISTANT CANCERS

In 2012, a report from the group first substantiated that the metastasis suppressor protein
NM23-H2/NME2 represses telomerase activity in cancer cells. Preliminary data towards
development of telomerase inhibiting peptide involved NME2-telomerase protein docking
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