FACT facilitates chromatin transcription by RNA polymerases I and III

Abstract

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, copurify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells

“…3,22,24,25 Our studies of SSRP1 and SPT16 mRNA and protein expression also support the notion that the two subunits act as a single unit: levels of the 2 subunits were found to be highly concordant on both the mRNA and protein levels in different cells, tissues, and conditions. 16 The work reported here shows that this concordance derives from complex reciprocal regulation between SSRP1 and SPT16 mRNAs and proteins.…”

“…These results indicate that neither FACT subunit mRNA is required for formation of a complex between the SSRP1 and SPT16 proteins, which is consistent with demonstration of SSRP1-SPT16 protein-protein binding in the absence of mRNAs in previous in vitro FACT complex reconstitution experiments. 3,20,21 Thus, we propose that SSRP1 mRNA is needed to maintain stability of the protein subunits of the complex in the cellular environment.…”

“…1 for review) and has been implicated in numerous biological processes that encompass chromatin. FACT assists in transcription driven by all three RNA polymerases, [1][2][3] replication, [4][5][6] recombination, 7 DNA damage responses, [7][8][9] and DNA methylation. 10 With respect to its role in regulation of gene expression, FACT is not a general enhancer of transcription, but appears to be specifically required for expression of particular genes that have highly ordered chromatin structure and are induced under certain conditions rather than constitutively expressed.…”

Basic concepts of epigenetics

“…150 While HP1α and HP1β are associated with silencing, other forms of HP1, such as HP1-type C, are found in euchromatin interacting with FACT subunit SSRP1, which is responsible for guiding FACT toward active genes to promote nucleosomal instability by H2AX exchange and RNAP activation. [103][104][105]151,152 Macromolecular Chromatin Euchromatin. At the macromolecular level, transcriptionally active euchromatin is housed at the inner nucleoplasm/center of chromosome territories, while silenced heterochromatin is arranged at the outer circular perimeter of the nuclear membrane.…”

“…102 FACT subunits facilitate phosphorylation of H2A, which is required for core exchange, as well as dimer ejection, nucleosomal collapse 103,104 and activation of RNAPs. 105 The function of FACT can be likened to a zipper effect that ejects nucleosomes along transcription start sites occupying unmethylated areas of DNA.…”