Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

Abstract

Metazoan replication-dependent (RD) histone genes encode the only known cellular mRNAs that are not polyadenylated. These mRNAs end instead in a conserved stem-loop, which is formed by an endonucleolytic cleavage of the pre-mRNA. The genes for all 5 histone proteins are clustered in all metazoans and coordinately regulated with high levels of expression during S phase. Production of histone mRNAs occurs in a nuclear body called the Histone Locus Body (HLB), a subdomain of the nucleus defined by a concentration of factors necessary for histone gene transcription and pre-mRNA processing. These factors include the scaffolding protein NPAT, essential for histone gene transcription, and FLASH and U7 snRNP, both essential for histone pre-mRNA processing. Histone gene expression is activated by Cyclin E/Cdk2-mediated phosphorylation of NPAT at the G1-S transition. The concentration of factors within the HLB couples transcription with pre-mRNA processing, enhancing the efficiency of histone mRNA biosynthesis.

Keywords: Cell cycle; Drosophila; histone genes; mRNA processing; nuclear body.

Figure 1.

Processing of histone pre-mRNA. (A) The histone pre-mRNA processing reaction. As the 3′ end of the histone mRNA is transcribed, SLBP binds to the stem-loop and the U7 snRNP binds to the HDE through base-pairing between the 5′ end of U7 snRNA and the Histone Downstream Element. This binding is stabilized by interaction of U7 snRNP with SLBP. The U7 snRNP consists of the U7 snRNA and 7 Sm proteins including Lsm10 and Lsm11, which replace SmD1 and SmD2 of the spliceosomal snRNPs. FLASH binds to Lsm11, and the FLASH/Lsm11 complex recruits the histone cleavage complex (HCC; blue) containing the Symplekin/CstF64 heterodimer and the CPSF73/CPSF100 heterodimer (dark blue). Cleavage of pre-mRNA is catalyzed by CPSF73. Other CPSF components are bound to the HCC but they may be sub-stoichiometric and not essential for processing (light blue). (B) U7 snRNP and FLASH are constitutive components of the HLB. In G1-phase they are either present separately or bound together in an inactive form. Processing is activated only during S phase after recruitment of the HCC by FLASH and U7 snRNP.

Figure 2.

Visualization of the HLB by immunofluorescence. (A) HeLa cell stained with antibodies recognizing Coilin (red) to detect Cajal bodies or FLASH (green) to detect HLBs. Note in this cell line these two bodies are distinct, although occasionally Coilin can be detected in the HLB (arrowhead). The number of HLBs in cultured cells depends on the ploidy. (B) HeLa cell stained with antibodies recognizing NPAT (red) or FLASH (green). Large HLBs correspond to the Hist1 cluster and smaller HLBs to the Hist2 cluster. Panels A and B were reproduced with permission from reference 42. (C) A Drosophila syncytial embryo stained with antibodies recognizing Mxc (red) and FLASH (green) and lamin (magenta; merged image). The chromosomes either are paired and nuclei contain one large HLB, or are separated and nuclei contain 2 smaller HLBs. (D) A Drosophila salivary gland cell stained with antibodies recognizing Mxc (red) and Lsm10 (green). Note that the HLB forms on the histone locus on polytene chromosomes. In all merge panels DNA is stained with DAPI (blue). © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the rightsholder.

Figure 3.

Schematic of Drosophila HLB assembly. The Drosophila replication dependent histone locus is organized as a tandem array of ∼100 copies of a 5 kb sequence containing one copy of each of the five RD histone genes. A sequence between the H3/H4 genes recruits Mxc and FLASH. Subsequent recruitment of other HLB components including U7 snRNP and Mute requires transcription initiation and results in maturation of a complete HLB. The result is a molecular environment that organizes the histone gene array and its accompanying trans-acting factors for efficient histone mRNA transcription and pre-mRNA processing.