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Project description:BackgroundCell type-specific use of cis-acting regulatory elements is mediated by the combinatorial activity of transcription factors involved in lineage determination and maintenance of cell identity. In macrophages, specific transcriptional programs are dictated by the transcription factor PU.1 that primes distal regulatory elements for macrophage identities and makes chromatin competent for activity of stimuli-dependent transcription factors. Although the advances in genome-wide approaches have elucidated the functions of these macrophage-specific distal regulatory elements in transcriptional responses, chromatin structures associated with PU.1 priming and the underlying mechanisms of action of these cis-acting sequences are not characterized.ResultsHere, we show that, in macrophages, FACT subunit SPT16 can bind to positioned nucleosomes directly flanking PU.1-bound sites at previously uncharacterized distal regulatory elements located near genes essential for macrophage development and functions. SPT16 can interact with the transcriptional co-regulator TRIM33 and binds to half of these sites in a TRIM33-dependent manner. Using the Atp1b3 locus as a model, we show that FACT binds to two positioned nucleosomes surrounding a TRIM33/PU.1-bound site in a region, located 35 kb upstream the Atp1b3 TSS, that interact with the Atp1b3 promoter. At this -?35 kb region, TRIM33 deficiency leads to FACT release, loss of the two positioned nucleosomes, RNA Pol II recruitment and bidirectional transcription. These modifications are associated with higher levels of FACT binding at the Atp1b3 promoter, an increase of RNA Pol II recruitment and an increased expression of Atp1b3 in Trim33-/- macrophages.ConclusionsThus, sequestering of SPT16/FACT by TRIM33 at PU.1-bound distal regions might represent a new regulatory mechanism for RNA Pol II recruitment and transcription output in macrophages.
| S-EPMC6647326 | biostudies-literature
Project description:Although the advances in genome-wide approaches have elucidated the functions of macrophage-specific distal regulatory elements in transcriptional responses, chromatin structures associated with PU.1 priming and the underlying mechanisms of action of these cis-acting sequences are not characterized. Here, we show that, in macrophages, FACT subunit SPT16 can bind to positioned nucleosomes directly flanking PU.1-bound sites at previously uncharacterized distal regulatory elements located near genes essential for macrophage development and functions. SPT16 can interact with the transcriptional co-regulator TRIM33 and binds to half of these sites in a TRIM33 dependent manner. Using the Atp1b3 locus as a model, we show that FACT binds to two positioned nucleosomes surrounding a TRIM33/PU.1-bound site in a region, located 35kb upstream the Atp1b3 TSS, that interact with the Atp1b3 promoter. At this -35kb region, TRIM33 deficiency leads to FACT release, loss of the two positioned nucleosomes, RNA Pol II recruitment and bidirectional transcription. These modifications are associated with higher levels of FACT binding at the Atp1b3 promoter, an increase of RNA Pol II recruitment and an increased expression of Atp1b3 in Trim33-/- macrophages. Thus, sequestering of SPT16/FACT by TRIM33 at PU.1-bound distal regions might represent a new regulatory mechanism for RNA Pol II recruitment and transcription output in macrophages.
2019-08-06 | GSE132922 | GEO
Project description:Although the advances in genome-wide approaches have elucidated the functions of macrophage-specific distal regulatory elements in transcriptional responses, chromatin structures associated with PU.1 priming and the underlying mechanisms of action of these cis-acting sequences are not characterized. Here, we show that, in macrophages, FACT subunit SPT16 can bind to positioned nucleosomes directly flanking PU.1-bound sites at previously uncharacterized distal regulatory elements located near genes essential for macrophage development and functions. SPT16 can interact with the transcriptional co-regulator TRIM33 and binds to half of these sites in a TRIM33 dependent manner. Using the Atp1b3 locus as a model, we show that FACT binds to two positioned nucleosomes surrounding a TRIM33/PU.1-bound site in a region, located 35kb upstream the Atp1b3 TSS, that interact with the Atp1b3 promoter. At this -35kb region, TRIM33 deficiency leads to FACT release, loss of the two positioned nucleosomes, RNA Pol II recruitment and bidirectional transcription. These modifications are associated with higher levels of FACT binding at the Atp1b3 promoter, an increase of RNA Pol II recruitment and an increased expression of Atp1b3 in Trim33-/- macrophages. Thus, sequestering of SPT16/FACT by TRIM33 at PU.1-bound distal regions might represent a new regulatory mechanism for RNA Pol II recruitment and transcription output in macrophages.
2019-08-06 | GSE117333 | GEO
Project description:Interplay between FACT subunit SPT16 and TRIM33 can remodel chromatin at macrophage distal regulatory elements
| PRJNA549472 | ENA
Project description:Interplay between FACT subunit SPT16 and TRIM33 can remodel chromatin at macrophage distal regulatory elements [ChIP-seq]
| PRJNA481772 | ENA
Project description:Interplay between FACT subunit SPT16 and TRIM33 can remodel chromatin at macrophage distal regulatory elements [ATAC-seq]
| PRJNA549476 | ENA
Project description:Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway.
| S-EPMC6486547 | biostudies-literature
Project description:The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.
| S-EPMC4741192 | biostudies-literature
Project description:The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-A crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.
| S-EPMC2449335 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2024-05-30 | GSE242380 | GEO