Page 71 - Biennial Report 2018-20 Jun 2021

generated in the laboratory, TRF2 occupancy was found on the hTERT promoter. Using HEK 293T,
MRC5 primary, HT1080 fibrosarcoma and HCT colon carcinoma cell lines, TRF2 occupancy at the
hTERT promoter was first established. Then, using promoter activity, mRNA expression, western
blot, immunofluorescence microscopy and telomerase activity assays it was established that
TRF2 is a negative regulator of hTERT, by observing increase in hTERT expression and telomerase
activity under TRF2-knockdown conditions. Changes in chromatin state were also studied by
looking at histone modifications and observed increase in H3K27Me3 suppression mark in the
hTERT promoter upon TRF2 knockdown. The interaction between TRF2 and REST repressor is
known and TRF2 was also observed to recruit REST at the p21 promoter to suppress its
expression in the lab. In this case also, occupancy of REST in the hTERT promoter was observed.

GENOME ENGINEERING TO GENERATE CELL AND ANIMAL MODELS TO
DELINEATE MELANOCYTE CELL FATE DECISIONS

In this reporting period, a CRISPR mutant of pigmentation relevant genes Carbonic Anhydrase 14
(CA14) in zebrafish was generated. CA14 has been identified in the laboratory as a key lead
candidate that could mediate melanocyte maturation by activating the MITF transcription factor
network. A germline mutant in zebrafish was created by targeting the ca14 coding region using
the CRISPR-Cas9 system. Cas9 - guide RNA complex was injected to fertilized zebrafish embryos
at the single cell stage. A variety of phenotypes in F0 were observed, which include microcephaly,
small eye size, mild enlargement of heart and decreased pigmentation. These phenotypes
recapitulate the known high expression of Ca14 in the brain, heart and eye. After screening for
mutants using T7 endonuclease assay, siblings were grown to adulthood. Genotyping revealed a
frame-shift mutation at the third codon by the deletion of two bases. Thereby, the mutant gene
would encode a truncated protein lacking most of the coding amino acids. Further experiments
were carried out using F2 embryos obtained from the in-cross of a homozygous mutant male
and a heterozygous female fish with the same mutation. Around 50% of embryos were obtained
with a smaller eye size and decreased pigmentation, following a Mendelian pattern of
inheritance. Mutant embryos showed the two base deletion and the non-phenotypic siblings
were heterozygous based on PCR confirmation of the mutation. Thereby, the pigmentation
phenotype observed in the morphant embryos is recapitulated in the genetic mutants, hence
ascertaining the regulatory role of Ca14 in the maturation of melanocytes.

In the adult stage, Ca14 mutation had a visible decrease in pigmentation, however high
melanophore density present in the lateral and dorsal region precluded assessment of melanin.
The wild type adult and the ca14fs003-/- fishes were subjected to dark adaptation to disperse
the existing melanin within the melanocyte, so that the content could be easily assessed. Distinct
non-overlapping melanophores present in fourth and fifth stripes near the caudal fin were
observed and, in this region, a substantial reduction in the melanin content could be observed
in the mutant fish.

The expression of differentiation genes when the pigment cells undergo migration and
maturation was studied. A decreased gene expression of tyr, tyrp1b and dct could be observed
in the mutant embryos; confirming that the cells were in an immature less pigmented state and
the pigmentation promoting gene expression was severely reduced in the absence of Ca14. The
wild type and the ca14fs003-/- were then subjected to intracellular pH measurements. Mutant

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