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1.
Nat Commun ; 15(1): 4450, 2024 May 24.
Article in English | MEDLINE | ID: mdl-38789411

ABSTRACT

Histone deacetylases (HDACs) play a crucial role in transcriptional regulation and are implicated in various diseases, including cancer. They are involved in histone tail deacetylation and canonically linked to transcriptional repression. Previous studies suggested that HDAC recruitment to cell-cycle gene promoters via the retinoblastoma (RB) protein or the DREAM complex through SIN3B is essential for G1/S and G2/M gene repression during cell-cycle arrest and exit. Here we investigate the interplay among DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. Knockout of SIN3B does not globally derepress cell-cycle genes in non-proliferating HCT116 and C2C12 cells. Loss of SIN3A/B moderately upregulates several cell-cycle genes in HCT116 cells but does so independently of DREAM/RB. HDAC inhibition does not induce general upregulation of RB/DREAM target genes in arrested transformed or non-transformed cells. Our findings suggest that E2F:RB and DREAM complexes can repress cell-cycle genes without relying on HDAC activity.


Subject(s)
E2F Transcription Factors , Histone Deacetylases , Repressor Proteins , Retinoblastoma Protein , Humans , Histone Deacetylases/metabolism , Histone Deacetylases/genetics , HCT116 Cells , Repressor Proteins/metabolism , Repressor Proteins/genetics , E2F Transcription Factors/metabolism , E2F Transcription Factors/genetics , Retinoblastoma Protein/metabolism , Retinoblastoma Protein/genetics , Mice , Animals , Sin3 Histone Deacetylase and Corepressor Complex/metabolism , Sin3 Histone Deacetylase and Corepressor Complex/genetics , Kv Channel-Interacting Proteins/metabolism , Kv Channel-Interacting Proteins/genetics , Cell Cycle/genetics , Promoter Regions, Genetic/genetics , Gene Expression Regulation , Genes, cdc
2.
Front Mol Biosci ; 9: 1040106, 2022.
Article in English | MEDLINE | ID: mdl-36387287

ABSTRACT

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through ß-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

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