Page 92 - Annual report 2021-22

Annual Report 2021-22 |

migration in cancerous conditions such as melanoma and in repopulating melanocytes in degenerative
disorders such as vitiligo.

Using cell-based and zebrafish model systems, Vivek’s group elucidated a pH mediated feed-forward
mechanism of epigenetic regulation that enables selective amplification of melanocyte maturation
program. They demonstrate that MITF activation directly elevates the expression of Carbonic
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Anhydrase 14 (Ca14) enzyme. Nuclear localized Ca14 increases the intracellular pH, resulting in the
activation of histone acetyl transferase activity of p300/CBP. In turn enhanced H3K27 histone
acetylation marks of select differentiation genes facilitates their amplified expression by MITF. CRISPR-
mediated targeted missense mutation of CA14 in zebrafish results in immature acidic melanocytes
with decreased pigmentation, establishing the centrality of this mechanism in rapidly activating
melanocyte differentiation. This work revealed a novel epigenetic control through pH modulation that
reinforces a deterministic cell fate by altering chromatin dynamics.

Under this project the group has been able to successfully establish the CRISPR based mutagenesis for
creating targeted mutagenesis in zebrafish model system. They have developed several mutant lines
from which the carbonic anhydrase knockout Ca14, H2A.Z.2 emerged as a complete knockout which
has now been extensively characterized for its role in melanocyte specification and maturation
process respectively.

Modeling skin pigmentation and development in stem cells

One of the aims of T. N. Vivek’s laboratory is to develop methods to achieve target cell regeneration
in skin. In this direction, studies on directed differentiation of cells to melanocyte, hepatocyte and
neuronal lineage using defined factors and model appropriate diseases in them. Melanocyte
generation from iPSC and characterization of the derived intermediates has been established. Single
cell sequencing of cultured melanocytes identified a defined subset of cells that indicate altered
maturation status and proliferative population being present in the same population. Somatic
mutations in vitiligo indicate lower burden of mutations in the matched lesional compared to the non-
lesional skin.

On the trail of an exonuclease

ExoN belongs to DEDDh family of exonucleases present across all kingdoms of life including DNA
viruses, and constitute enzymes involved in DNA proof-reading as well as RNA metabolism. Multiple
sequence alignment of ExoN with diverse homologs suggested conservation of core catalytic motif
involved in exonuclease activity. Two zinc finger (ZnF) like motifs were observed within this enzyme
that are unique to nidovirales, and the crystal structure of SARS-CoV ExoN confirms the co-ordination
of zinc by these two motifs. Interestingly, two of the core catalytic residues fall within the second zinc
finger motif (ZnF2). Hence ExoN is structurally and possibly functionally distinct from other DEDDh
enzymes, whose origin in RNA viruses is enigmatic. To identify the common recent ancestor of ExoN,
position specific iterative-blast was carried out using ExoN from members of tobaniviridae.
Convergence of iteration led us to a cluster of potential hits from cellular organisms. Interestingly,
eukaryotic antiviral protein NFX1 type zinc finger containing protein (ZNFX1) emerged as the candidate
with substantial similarity to viral ExoN.

Vivek’s group performed sequence based identification of recent common ancestors to identify the
evolutionary origin of this enzyme family in coronaviruses. Upon bioinformatic analysis they inferred