Identification of PAX6 and NFAT4 as the Transcriptional Regulators of the Long Noncoding RNA Mrhl in Neuronal Progenitors

ABSTRACT

The long noncoding RNA (lncRNA) Mrhl has been shown to be involved in coordinating meiotic commitment of mouse spermatogonial progenitors and differentiation events in mouse embryonic stem cells. Here, we characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid-derived radial-glia-like neuronal progenitor cells. Depletion of Mrhl leads to early differentiation of neuronal progenitors to a more committed state. A master transcription factor, PAX6, directly binds to the Mrhl promoter at a major site in the distal promoter, located at 2.9 kb upstream of the transcription start site (TSS) of Mrhl. Furthermore, NFAT4 occupies the Mrhl-proximal promoter at two sites, at 437 base pairs (bp) and 143 bp upstream of the TSS. Independent knockdown studies for PAX6 and NFAT4 confirm that they regulate Mrhl expression in neuronal progenitors. We also show that PAX6 and NFAT4 associate with each other in the same chromatin complex. NFAT4 occupies the Mrhl promoter in PAX6-bound chromatin, implying possible coregulation of Mrhl. Our studies are crucial for understanding how lncRNAs are regulated by major lineage-specific transcription factors, in order to define specific development and differentiation events.

KEYWORDS:

• Mrhl
• lncRNA
• NPCs
• transcription factor
• brain
• PAX6

Declaration of Interests

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

We thank Suma B. Srinivas of the Confocal Imaging Facility, Prakash R. Gnaneshwar of the Animal Facility, and Anitha S. Rokhade of the Sequencing Facility at JNCASR, India.

M.R.S.R. acknowledges the Department of Science and Technology, Government of India, for a SERB Distinguished Fellowship and SERB-YOS (Year of Science Chair Professorship). D.P. thanks the University Grants Commission, Government of India, and JNCASR, India, for her Ph.D. fellowship. S.D. thanks the Department of Biotechnology, Government of India, for her postdoctoral fellowship. This work was financially supported by the Department of Biotechnology, Government of India (grant numbers BT/01/COE/07/09 and DBT/INF/22/SP27679/2018).

We declare that we have no competing interests.

Conceptualization: M.R.S.R., D.P. Methodology: D.P., S.D., D.P.I., D.S., M.R.S.R. Software: U.B. Validation: S.D., D.S. Formal Analysis: D.P., S.D., D.P.I., U.B. Investigation: D.P., S.D., D.P.I., U.B. Resources: M.R.S.R. Data curation: D.P., U.B. Writing - original draft and preparation: D.P., S.D. Writing - review and editing: M.R.S.R. Visualization: D.P., M.R.S.R. Supervision: M.R.S.R. Project administration: M.R.S.R. Funding acquisition: M.R.S.R.