FACT (biology)
Encyclopedia
FACT is a heterodimeric protein complex that affects eukaryotic RNA polymerase II
(Pol II) transcription elongation both in vitro and in vivo. It was discovered in 1998 as a factor purified from human cells, consisting of 140 and 80 kilodalton (kDa) subunits, that was essential for productive in vitro Pol II transcription on a chromatinized DNA template. The 140 kDa subunit is encoded by a human gene (SUPT16H
) which is 36% identical to the S. cerevisiae gene Spt16 and the 80 kDa subunit is human SSRP1
(POB3 in S. cerevisiae). Both of these subunits in yeast affect Pol II transcription elongation, and purified human FACT binds specifically to mononulceosomes and the histone H2A/H2B dimer, but not to the H3/H4 tetramer (see: Nucleosome core particle) or Pol II. Co-immunoprecipitation assays with tagged recombinant proteins showed that the Spt16 subunit interacts with H2A/H2B dimers and mononucleosomes, but not H3/H4 tetramers, whereas the SSRP1 subunit interacts only with H3/H4 tetramers and not mononucleosomes. Deletion of the highly acidic carboxy-terminus of Spt16 (a common feature of known histone chaperones) does not prevent Spt16 from forming a stable complex with SSRP1, but it does eliminate interaction with mononucleosomes and ability to stimulate in vitro transcription on chromatinized templates. The two subunits together, but neither alone, can stimulate formation of nucleosomes from free histones and DNA (histone chaperone activity). These two subunits are highly conserved across all eukaryotes and in addition to transcription, have been shown to affect DNA repair and replication as well.
In cells, FACT is enriched on parts of the genome involved in actively elongating Pol II, as seen in fluorescent-antibody staining of Drosophila
polytene chromosomes
and chromatin immunoprecipitation
(ChIP) assays on Drosophila Kc cell extracts.
RNA polymerase II
RNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
(Pol II) transcription elongation both in vitro and in vivo. It was discovered in 1998 as a factor purified from human cells, consisting of 140 and 80 kilodalton (kDa) subunits, that was essential for productive in vitro Pol II transcription on a chromatinized DNA template. The 140 kDa subunit is encoded by a human gene (SUPT16H
SUPT16H
FACT complex subunit SPT16 is a protein that in humans is encoded by the SUPT16H gene.-Further reading:...
) which is 36% identical to the S. cerevisiae gene Spt16 and the 80 kDa subunit is human SSRP1
Structure specific recognition protein 1
Structure specific recognition protein 1, also known as SSRP1, is a human protein.-Interactions:Structure specific recognition protein 1 has been shown to interact with NEK9....
(POB3 in S. cerevisiae). Both of these subunits in yeast affect Pol II transcription elongation, and purified human FACT binds specifically to mononulceosomes and the histone H2A/H2B dimer, but not to the H3/H4 tetramer (see: Nucleosome core particle) or Pol II. Co-immunoprecipitation assays with tagged recombinant proteins showed that the Spt16 subunit interacts with H2A/H2B dimers and mononucleosomes, but not H3/H4 tetramers, whereas the SSRP1 subunit interacts only with H3/H4 tetramers and not mononucleosomes. Deletion of the highly acidic carboxy-terminus of Spt16 (a common feature of known histone chaperones) does not prevent Spt16 from forming a stable complex with SSRP1, but it does eliminate interaction with mononucleosomes and ability to stimulate in vitro transcription on chromatinized templates. The two subunits together, but neither alone, can stimulate formation of nucleosomes from free histones and DNA (histone chaperone activity). These two subunits are highly conserved across all eukaryotes and in addition to transcription, have been shown to affect DNA repair and replication as well.
In cells, FACT is enriched on parts of the genome involved in actively elongating Pol II, as seen in fluorescent-antibody staining of Drosophila
Drosophila
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
polytene chromosomes
Polytene chromosome
To increase cell volume, some specialized cells undergo repeated rounds of DNA replication without cell division , forming a giant polytene chromosome...
and chromatin immunoprecipitation
Chromatin immunoprecipitation
Chromatin Immunoprecipitation is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or...
(ChIP) assays on Drosophila Kc cell extracts.