The mouse Supt16h/Fact140 gene, encoding part of the FACT chromatin transcription complex, maps close to Tcra and is highly expressed in thymus.

Chromatin remodeling complexes are important factors in the regulation of RNA transcription, DNA replication, recombination, and repair. Two major types of chromatin remodeling enzymes appear to be highly conserved: the ATP-dependent remodeling complexes and nuclear histone acetyltransferases (HATs) and deacetylases (HDACs). Recently another chromatin remodeling complex, “facilitates chromatin transcription” (FACT), was isolated and described as a complex that can facilitate RNA polymerase II elongation through nucleosomes (Orphanides et al. 1999). In this report we describe the cloning, mapping, and expression of Supt16h, the mouse homolog of human FACT140 and yeast Cdc68/Spt16. Chromatin remodeling and covalent modification of histones are important in transcriptional regulation of chromatin templates (Kingston and Narlikar 1999). Remodeling complexes, such as yeast Swi/Snf, affect transcription positively by allowing sequence-specific activation factors to bind to their sites in the chromatin and to allow RNA polymerase access to the gene. Others, such as HATs and HDACs, interact with sequence-specific transcription factors, causing local hyperor hypoacetylation of histones and therefore allowing transcription and/or repression of specific genes. HATs/HDACs and Swi/Snf components can interact and may work cooperatively. Another important factor involved in chromatin remodeling is the recently described elongation factor FACT (Orphanides et al. 1999). FACT interacts with nucleosomes and histone H2A/H2B dimers, suggesting that it may work by promoting nucleosome disassembly upon transcription (Orphanides et al. 1999). There is an equivalent complex known from Xenopus: the DNA unwinding factor (DUF), and from yeast: the CP complex (Okuhara et al. 1999; Brewster et al. 1998). These complexes consists of an HMG-box containing protein component of ∼80 kDa and a ∼140-kDa protein component. The HMG-box protein in humans is known as structure-specific recognition protein-1 (SSRP1; Bruhn et al. 1992). Other HMG-box protein homologs have been found throughout the eukaryotes, including the mouse T160 protein (Shirakata et al. 1991), the Xenopus DUF80 (Okuhara et al. 1999), and the yeast Pob3 (the yeast homologs lack the HMG box sequence) (Wittmeyer and Formosa 1997). The larger component of FACT is also found throughout the eukaryotes and is known as FACT140 in human, DUF140 in Xenopus, and Cdc68 or Spt16 in the prototypical yeast gene. The CP complex aids transcription of a number of genes and facilitates repression at promoters lacking DNA-bound trans-activators (Malone et al. 1991). DUF is involved in DNA replication through unwinding of duplex DNA (Okuhara et al. 1999). T cell development in the thymus requires the coordinated expression of thymus-specific and ubiquitously expressed genes and recombination of T cell receptor (TCR) genes, all of which require chromatin remodeling (Kim et al. 1999; Cherry and Baltimore 1999). The mouse T160 protein (homolog of SSRP1, DUF80, and Pob3) was originally cloned by selection of proteins binding to V(D)J recombination signal sequences (Shirakata et al. 1991), although further work has shown that it binds to cruciform or linear duplex DNA without apparent sequence specificity, bending the DNA in the process (Gariglio et al. 1997). Thus, it is likely that protein complexes involved in chromatin remodeling and transcription have an important role in T cell development and possibly in V(D)J rearrangement. We have investigated differential gene expression in developing mouse thymocytes at the stage known as “b-selection,” when the pre-TCR is expressed on the cell surface and the thymocytes differentiate from CD48 cells to CD48, undergoing massive expansion in number. We created a subtraction cDNA library from TCR a thymus (C57BL/6J-Tcra, Jackson Laboratory, Bar Harbor, Me.) subtracted with RAG-1 thymus (C57BL/6JRag1). The sequences of 67 randomly picked clones were analyzed and used to search the GenBank, EMBL, DDBJ, and PDB databases by using the BLASTN program (Altschul et al. 1997). A majority of the clones isolated and identified carried out some general function that is required by growing and proliferating cells, including translation factors and transcription factors, DNA binding proteins, and proteins involved in signal transduction, cytokinesis, energy production, and molecular motors. One clone of particular interest was the mouse homolog of yeast Cdc68/Spt16 and human FACT140. The Fact140 cDNA contains an ORF of 3.1 kb, which encodes for a protein of 1047 amino acids, predicted mw 119,726 Da and pI of 5.42. Fact140 is a highly charged protein, like its homolog in yeast and humans, containing 17.5% acidic and 14.7% basic residues. Alignment of Fact140 with related sequences from plants to yeast to mammals shows a great deal of conservation across species (Fig. 1). Fact140 shares 80% identity with vertebrate proteins DUF140 and FACT140 and 33% and 35% identity with yeast Cdc68/Spt16 and Arabidopsis thaliana sequences respectively. All FACT140 orthologous proteins share a highly conserved acidic carboxyl terminus rich in glutamic and aspartic acid residues (Fig. 1). There is also a highly conserved bipartite nuclear localization signal sequence (residues 491–507 of mouse Fact140). There are numerous potential phosphorylation sites throughout the protein on Ser, Thr, and/or Tyr residues. Fact140 appears to be expressed in all murine tissues and cultured cells examined by Northern blot GenBank accession #: AF323667