Int J Clin Exp Pathol 2017;10(5):5334-5342 www.ijcep.com /ISSN:1936-2625/IJCEP0047442 Original Article Characterization of the N-terminally clipped histone H3 (∆H3) from old chicken and rat liver Neetika Singh 1 , Jogeswar S Purohit 1,2 , S Shanti 1 , Abhilasha Singh 1 , Anil K Panigrahi 1,3 , Madan M Chaturvedi 1 1 Laboratory for Chromatin Biology, Department of Zoology, University of Delhi, North Campus, Delhi 110007, India; 2 Cluster Innovation Centre, G. C. Narang Marg, University of Delhi, North Campus, Delhi 110007, India; 3 Present Address: Department of Pediatric Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA Received December 27, 2016; Accepted February 14, 2017; Epub May 1, 2017; Published May 15, 2017 Abstract: Site-specifc proteolysis of the N- or C- terminus of histone tails has recently emerged as a novel form of irreversible post-translational modifcations of histones. A proteolytically cleaved H3 product (named as ∆H3) has recently been reported in the liver of old chicken. In the present study, a comparison of the N-terminal amino acid sequences of the H3 and the ∆H3 revealed that the ∆H3 lacked 23 amino acids from the N-terminus. The proteoly- sis was observed only in histone H3 in the liver of old chicken, and it was absent in brain and erythrocytes. A ∆H3 like product was also observed in the liver of old rats. Further, the ∆H3 could be probed with H3 specifc N-terminal pan acetylated antibody, suggesting that the chromatin domains containing the ∆H3 would be transcriptionally repressive. However, the ∆H3 could be probed with anti-H3K36me2 antibody (broadly a mark of transcriptionally permissive chromatin), suggesting that the ∆H3 though lacked major portion of the N-terminal region, still be impor- tant in determining the transcriptional status of the chromatin. Therefore, a comparison of the existing epigenetic marks on H3 and ∆H3 is also tabulated in the present study. To ascertain whether the cleavage is dependent on post-translational modifcations of histone H3, bacterially expressed recombinant H3 was used as substrate for in vitro cleavage reaction. The analyses of the products revealed that the major cleaved product was different from that of the ∆H3, suggesting that the in vivo generation of ∆H3 requires histone H3 to be chromatin-bound. Keywords: Histone H3, histone H3 proteolysis, ∆H3, epigenetic marks Introduction The N- and the C- terminal tails of core histones are subjected to a diverse array of reversible post-translational modifcations. Some of the widely-studied modifcations are methylation, acetylation, phosphorylation, ubiquitylation and ADP-ribosylation etc [1]. In addition to these reversible modifcations, histones also undergo irreversible modifcations by proteoly- sis of the N- or the C- terminal of histone tails, which also have been correlated to epigenetic regulation [2-9]. Proteolysis of the N- or the C- terminal histone tails have been reported in Tetrahymena [2], mammalian kidney cells (BHK cells) infected with Foot and Mouth Disease Virus (FMDV) [10, 11], cycad pollen [12], rat uterus [13], yeast [14, 15], mouse embryonic stem cells [16], human embryonic stem cells [17], calf thymus [18], monocytes [19] and chicken liver [20]. A recent attempt has been made to divide his- tone proteolysis into histone degradation and epigenetically inferential tail clipping [21]. Recently, our group has shown that chicken liver nuclear glutamate dehydrogenase (GDH) cleaves histone H3 in vitro [22]. By sequencing of the clipped H3 products, the in vitro proteo- lytic cleavage sites by GDH have been mapped at R2/T3, K4/Q5, R8/K9, G13/K14, K23/A24 and K27/S28 of histone H3 [22, 23]. Further, in the liver of old chicken, the histone H3 was N-terminally clipped in vivo and the product was named as ∆H3 [23, 24]. Another group has mapped the cleavage site at R26/K27 of his- tone H3 [24]. However, a careful analysis of the