Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions

Abstract

SynopsisThe BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted HisBcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser 250 , which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser 250 substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser 250 phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys 268 in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.

The human Cranio Facial Development Protein 1 (Cfdp1) gene encodes a protein required for the maintenance of higher-order chromatin organization

“…Cfdp1 is 139,815 bp long with 7 exons and 6 introns and maps to chromosome 16 in the band 16q22.2-q22.3, where it is proximally and distally flanked by Bcar1 and Tmem170A genes, respectively12131415. Cfdp1 expression has been detected in a wide range of human tissues, including cancer tissues.…”

“…Cfdp1 expression has been detected in a wide range of human tissues, including cancer tissues. It encodes a protein of 299 amino acids, called CFDP1, belonging to the evolutionarily conserved family of Bucentaur (BCNT) proteins12131415. The CFDP1 protein is characterized by an 82-amino acid region located at the C-terminus, called the BCNT-C domain, which is highly conserved among almost all eukaryotes, while the N-terminal region is more divergent121314.…”

Assessment of a western blot signal for the Bcnt/Cfdp1, a tentative component of Srcap chromatin remodeling complex; trial to overcome off-target problems

“…We identified the Bcnt (Bucentaur) / Cfdp1 (craniofacial developmental protein 1) protein family, which probably function as a component of chromatin remodeling complex by the study of yeast ortholog Swc5 (11). Previously, we raised an Ab against a peptide of a C-terminal region of BCNT (called BCNT-C), EELAIHNRGKEGYIERKA, which is evolutionarily conserved from yeast to humans (12).…”

“…Culture of E. coli and induction of coded molecules by cDNAs in pCold II vector were described previously (11). E. coli were harvested and washed with PBS by centrifugation at 5,000 x g for 5 min.…”

The role of JNK phosphorylation as a molecular target to enhance adenovirus replication, oncolysis and cancer therapeutic efficacy

“…8,13,19 Mechanistically, JNK induces autophagy via the phosphorylation of BCL-2 to overcoming the inhibition of Beclin-1 via un-phosphorylated BCL-2 in the cytosol which freely forms an inhibitory complex with Beclin-1. 13,[19][20][21][22][23][24] JNK and the map kinase (MAPK) pathway also play a crucial role to upregulate ATG5 expression. 25 JNK1 and JNK2 are also known known as MAPK8 and MAPK9 respectively.…”