Alshehri, A. | Search bioRxiv

bioRxiv2025

Mutations on the surface of HDAC1 reveal molecular determinants of specific complex assembly and their requirement for gene regulation

Histone deacetylase 1 and 2 (HDAC1/2) are highly related enzymes that regulate histone acetylation levels in all cells, as catalytic and structural components of six unique multiprotein complexes: SIN3, NuRD, CoREST, MIDAC, MIER and RERE. Co-immunoprecipitation of HDAC1-Flag followed by mass spectrometry revealed that 92% of HDAC1 in mouse embryonic stem cells resides in 3 complexes, NuRD (49%), CoREST (28%) and SIN3 (15%). We compared the structures of MTA1:HDAC1 and MIDAC:HDAC1 to identify critical binding residues on the surface of HDAC1. Surprisingly, a single mutation, Y48E, disrupts binding to all complexes except SIN3. Rescue experiments performed with HDAC1-Y48E in HDAC1/2 double-knockout cells, showed that retention of SIN3 binding alone is sufficient for cell viability. Gene expression and histone acetylation patterns were perturbed in both Y48E and a second mutant cell line, HDAC1-E63R, indicating that cells require a full repertoire of the HDAC1/2 complexes to regulate their transcriptome appropriately. Comparative analysis of MTA1/HDAC1 and SIN3B/HDAC2 structures confirmed the differential modes of HDAC1 recruitment, such that Y48 interacts with ELM2/SANT domain-containing proteins, but not SIN3. The E63R mutation shows markedly reduced binding to NuRD and MiDAC complexes, but retains some CoREST binding. We provide novel molecular insights into the abundance, co-factors and assemblies of this crucial family of chromatin modifying machines.

molecular biology↗

bioRxiv2024

SARS-CoV-2 S, M and E Structural Proteins Down-modulate HIV-1 LTR Activity and Modulate Endoplasmic Reticulum Stress Responses

We have previously shown that the Hepatitis C Virus (HCV) E1E2 envelope glycoprotein can down-modulate HIV-1 long-terminal repeat (LTR) activity through disruption to NF-{kappa}B activation. This response is associated with up-regulation of the endoplasmic reticulum (ER) stress response pathway. Here we demonstrate that the SARS-CoV-2 S, M and E but not the N structural protein can perform similar down-modulation of HIV-1 LTR activation and in a dose-dependent manner in both HEK293 and lung BEAS-2B cell-lines and interpreted as a result of NF-{kappa}B down-modulation. The effect is highest with the SARS-CoV-2 Wuhan S strain and decreases over-time for the subsequent emerging variants of concern (VOC) with omicron providing the weakest effect. We developed pseudo-typed viral particle (PVP) molecular viral tools that allowed for the generation of cell-lines constitutively expressing separately the four SARS-CoV-2 structural proteins and utilising the VSV-g envelope protein to deliver the integrated gene construct. Differential gene expression analysis (DGEA) was performed on cells expressing S, E, M or N to determine cell activation status. It it was determined that gene expression differences were found in a number of interferon-stimulated genes (ISGs), including IF16, IFIT1, IFIT2 and ISG15 as well as for a number of heat shock protein (HSP) genes, including HSPH1, HSPA6 and HSPBP1 with all four SARS-CoV-2 structural proteins. There were also differences observed with expression patterns of transcription factors with both SP1 and MAVS upregulated in the presence of S, M and E but not the N protein. Collectively the results indicate that gene expression patterns associating with ER stress pathways can be identified with SARS-CoV-2 envelope glycoprotein expression. The results suggest the SARS-CoV-2 can modulate activation of an array of cell pathways resulting in disruption to NF-{kappa}B signalling hence providing alterations to multiple physiological responses of SARS-CoV-2 infected cells.

immunology↗