Abstract
The conserved and essential histone chaperone, facilitates chromatin transcription (FACT), reorganizes nucleosomes during DNA transcription, replication, and repair and ensures both efficient elongation of RNA Pol II and nucleosome integrity. In mammalian cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in contrast to yeast, FACT accumulates at the transcription start site of genes reminiscent of RNA polymerase II profile. Depletion of FACT in mouse embryonic stem cells leads to deregulation of developmental and pro-proliferative genes concomitant with hyper-proliferation of mES cells. Using MNase-seq, Assay for Transposase-Accessible Chromatin sequencing, and nascent elongating transcript sequencing, we show that up-regulation of genes coincides with loss of nucleosomes upstream of the transcription start site and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to regulate the expression of these genes. Finally, we demonstrate a role for FACT in cell fate determination and show that FACT depletion primes embryonic stem cells for the neuronal lineage.
Highlights
The basic functional unit of chromatin is the nucleosome consisting of around 147 bp of DNA wrapped around an octamer of histone proteins—two copies each of histones H2A, H2B, H3, and H4
We have addressed the role of the histone chaperone facilitates chromatin transcription (FACT) in mouse embryonic stem (ES) cells
In contrast to the genomic profile identified for Saccharomyces cerevisiae FACT, where the protein occupancy is depleted at the TSS and accumulates in the gene body (True et al, 2016), the genomic profile of mammalian FACT over active genes is reminiscent of a profile of the Ser5 phosphorylated form of RNA Pol II
Summary
The basic functional unit of chromatin is the nucleosome consisting of around 147 bp of DNA wrapped around an octamer of histone proteins—two copies each of histones H2A, H2B, H3, and H4. In vitro, chromatinized DNA templates are refractory to transcription, suggesting that the nucleosome might provide a barrier for the elongating RNA polymerase. It has been demonstrated that FACT can cooperate with all RNA polymerases in the cell and ensure both efficient transcription elongation and nucleosome integrity. Both FACT subunits are highly conserved across all eukaryotes with the exception of an HMG-like domain present in SSRP1 but absent in the yeast homolog Pob. An HMG domain protein named Nhp has been proposed to provide the DNA binding capacity of FACT (Formosa et al, 2001)
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