Abstract
FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT and YARP–NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single -helix that makes multiple contacts with -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.
Highlights
Expression of replication-dependent histone genes is restricted to S-phase of the cell cycle, occurring concomitantly with DNA replication and providing a bulk of histone proteins for de novo synthesized DNA [1,2,3,4]
Previous results indicated that the C-terminal fragments of FLICE-associated huge protein (FLASH) and Yang 1-Associated Protein-Related Protein (YARP) fold into three α-helices, structurally resembling the DNA binding domain of the Myb oncogene and the SANT domain found in several chromatin-remodeling and modifying complexes [30]
To characterize these two domains in more detail, we expressed the C-terminal region of FLASH and the corresponding region of YARP in bacteria and analyzed their structure by NMR spectroscopy
Summary
Expression of replication-dependent histone genes is restricted to S-phase of the cell cycle, occurring concomitantly with DNA replication and providing a bulk of histone proteins for de novo synthesized DNA [1,2,3,4]. Transcriptional activity of all classes of replication-dependent histone genes is controlled by Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT). At the G1/S phase transition, this 200 kDa protein is phosphorylated at multiple sites by a complex of cyclin E and cyclin dependent kinase 2 (CDK2), coinciding with the activation of histone gene expression [6,7,8]. Hyper-phosphorylation of pRb liberates E2F, which activates transcription of a subset of genes that encode proteins required to support DNA replication [12,13]. The synchronized phosphorylation of NPAT and pRb by the CDK2/cyclin E complex is important for timely synthesis of the two key chromatin components during the S phase: DNA and histone proteins
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