Camello, a novel family of Histone Acetyltransferases that acetylate histone H4 and is essential for zebrafish development

OPEN SUBJECT AREAS: ACETYLATION DATA MINING Received 10 February 2014 Accepted 7 July 2014 Published 15 August 2014 Correspondence and requests for materials should be addressed to K.K. (krish@iiserpune. ac.in) * Current address: Persistent Systems Camello, a novel family of Histone Acetyltransferases that acetylate histone H4 and is essential for zebrafish development Krishanpal Karmodiya, Krishanpal Anamika*, Vijaykumar Muley, Saurabh J. Pradhan, Yoshita Bhide & Sanjeev Galande Center of Excellence in Epigenetics, Indian Institute of Science Education and Research, Pashan, Pune 411 021, India. In this study, we have investigated genome-wide occurrence of Histone Acetyltransferases (HATs) in genomes of Mus musculus and Danio rerio on the basis of presence of HAT domain. Our study identified a group of proteins that lacks characteristic features of known HAT families, relatively smaller in size and has no other associated domains. Most of the proteins in this unclassified group are Camello proteins, which are not yet known and classified as functional HATs. Our in vitro and in vivo analysis revealed that Camello family proteins are active HATs and exhibit specificity towards histone H4. Interestingly, Camello proteins are among the first identified HATs showing perinuclear localization. Moreover, Camello proteins are evolutionarily conserved in all chordates and are observed for the first time in cnidarians in phylogeny. Furthermore, knockdown of Camello protein (CMLO3) in zebrafish embryos exhibited defects in axis elongation and head formation. Thus, our study identified a novel family of active HATs that is specific for histone H4 acetylation, exhibits perinuclear localization and is essential for zebrafish development. Limited, Pingala Aryabhata, Erandwane, Pune 411004, India. G ene expression in eukaryotes is a tightly controlled process involving a complex interplay between chromatin proteins and transcription factors. The functional availability of these factors and accessibility of DNA sequence define the state of gene activation or repression. DNA in chromatin is wrapped around histone octamers comprising of two copies each of the four core histone proteins (H2A, H2B, H3 and H4) to form discrete nucleosome units. The N-terminal tails of these core-histones protrude from the nucleosome particles and are subjected to various post-translational modifications such as acetylation, methylation, phosphorylation and ubiqutination1,2. Histone acetylation by histone acetyltransferases (HATs) is one of the most extensively studied covalent histone modifications. HATs modify physico–chemical properties of core histones through acetylation, influence the nucleosome structure and participate in transcription regulation. However, many HATs can act on nonhistone proteins (cytoplasmic as well as nuclear) and are now renamed as lysine acetyltransferases (KATs)3. Acetylation of core-histone and non-histone proteins is correlated with various cellular processes such as transcription regulation, chromatin assembly, DNA repair and cell cycle progression4. Characterization of HATs on the basis of protein sequence and domain organization reveals five distinct families of HATs5. (i) Largest of these families is the GNAT (GCN5-related N-acetyltransferase) family whose members share a highly conserved acetylation-related structural motif. GCN5, one of the members of the GNAT family is the best-characterized HAT protein and serves as a prototype for histone acetyltransferase studies. One of the characteristic features of the GNAT family is a carboxy-terminal bromo-domain, which helps in targeting proteins to the substrate6. GNAT family proteins are also known to acetylate non-histone proteins as well as small molecules7. (ii) Another family is the MYST (MOZ, Ybf2/Sas3, Sas2 and Tip60) family, which also has an acetylation-related structural motif. Many of the MYST family proteins contain zinc fingers as well as chromo-domain5. Presence of chromo-domain in the MYST family suggests that they might interact with the heterochromatin-associated proteins8. GNAT and MYST families contain dozens of lysine acetyltransferase enzymes and are mostly part of multi-subunit transcriptional co-activator complexes. (iii) The P300/CBP (CREB-binding protein) family consists of two paralogous proteins, P300 and CBP. These two proteins have interchangeable functions. Members of the P300/CBP family contain many functional domains including SCIENTIFIC REPORTS | 4 : 6076 | DOI: 10.1038/srep06076 1 www.nature.com/scientificreports acetylation-related structural motif which is involved in acetyl-CoA binding, three zinc finger regions and a bromo-domain. P300/CBP act as co-activators and harbor domains for interaction with many transcription factors9. (iv) The fourth group of HATs is the basal transcription factor family, which is related to mammalian TAFII250, the largest subunit of the transcription factor complex TFIID2,10. Basal transcription factor family proteins also act as HATs but do not harbor acetylation related structural motif. (v) Last of the HAT families is the nuclear receptor cofactors family, which is largely specific to mammals6. Members of this family include nuclear receptor co-activators such as steroid receptor coactivators (SRC1) and clock circadian regulator (CLOCK). This family of HATs is also functionally known to act as HAT but they do not have any acetylation related structural motif11–13. Here, we performed genome-wide survey of lysine acetyltransferase proteins in mouse and zebrafish genomes. Our genome-wide bioinformatics analysis identified a novel family of HATs, namely Camello proteins, which harbors the HAT domain. We demonstrated that Camello-family of proteins are active HATs and have specificity towards histone H4 acetylation. We also show that Camello proteins have perinuclear localization and their overexpression leads to increased acetylation of histone H4. Finally, we demonstrated in vivo role of camello histone acetyltransferases by knockdown of CMLO3 in zebrafish embryos. Morpholino-mediated knockdown of CMLO3 exhibited defects in axis elongation and head formation, suggesting its critical role in zebrafish development. Results Genome-wide identification of HATs in mouse and zebrafish genomes. The mouse genome sequence was searched for homologs of known histone acetyltransferases. Briefly, we employed a query set of HATs from all kingdoms of life as proteins harboring known HAT domains and previously classified e.g. GCN5. A total of 293 HAT domain-containing proteins were identified from all kingdoms of life and their homologs were surveyed in the mouse proteome database. After removing redundant sequences and false positives, we obtained 33 putative HAT-like proteins in the mouse proteome. These 33 putative HAT-like proteins are encoded by 21 mouse genes indicating presence of isoforms for few of these proteins. Phylogenetic analysis of these 33 HATs reve (...truncated)