Biology subjects

Owen-Hughes, T.

Publications and source records attributed to Owen-Hughes, T..

5 recordsLinked to original sources

bioRxiv2023

Transcription promotes the restoration of chromatin following DNA replication.

DNA replication results in the transient eviction of nucleosomes, RNAPII and transcription regulators. How chromatin organization is duplicated on the two daughter strands is a central question in epigenetics. In mammals, transcription restarts on newly replicated DNA within a couple of hours, promoting chromatin accessibility. However, the role of transcription in the restoration of other chromatin determinants following DNA replication remains unclear. Here we have monitored protein re-association to newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that nucleosome assembly and the re-establishment of most histone modifications are uncoupled from transcription restart. However, upon transcription inhibition, the re-association of many proteins was altered, including ATP-dependent remodellers, transcription regulators, the histone variant H2A.Z, histone modifiers as well as the restoration of H3.3K36me2. Finally, transcription also provoked the recruitment of several DNA repair proteins, revealing that transcription promotes chromatin reestablishment post-replication but is also a potential source of genotoxic stress.

molecular biology↗

bioRxiv2020

SUMO maintains the chromatin environment of human induced pluripotent stem cells.

To determine the role of SUMO modification in the maintenance of pluripotent stem cells we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 initiated changes associated with loss of pluripotency such as reduced expression of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created 3 networks of proteins with functions in regulation of gene expression, ribosome biogenesis and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The remainder have roles in transcription and chromatin structure regulation. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centred on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO is involved in the maintenance of the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

cell biology↗

bioRxiv2018

Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome

ATP-dependent chromatin remodelling proteins represent a diverse family of proteins that share ATPase domains that are adapted to regulate protein-DNA interactions. Here we present structures of the yeast Chd1 protein engaged with nucleosomes in the presence of the transition state mimic ADP-beryllium fluoride. The path of DNA strands through the ATPase domains indicates the presence of contacts conserved with single strand translocases and additional contacts with both strands that are unique to Snf2 related proteins. The structure provides connectivity between rearrangement of ATPase lobes to a closed, nucleotide bound state and the sensing of linker DNA. Two turns of linker DNA are prised off the surface of the histone octamer as a result of Chd1 binding, and both the histone H3 tail and ubiquitin conjugated to lysine 120 are re-orientated towards the unravelled DNA. This indicates how changes to nucleosome structure can alter the way in which histone epitopes are presented.

molecular biology↗

bioRxiv2017

SINHCAF/FAM60A links SIN3A function to the hypoxia response and its levels are predictive of cancer patient survival

The SIN3A-HDAC complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF/FAM60A, links the SIN3A-HDAC co-repressor complex function to the hypoxia response. SINHCAF Chromatin Immunoprecipitation-sequencing and gene expression analysis reveal a signature associated with the activation of the hypoxia response. We show that SINHCAF specifically repress HIF 2 mRNA and protein expression resulting in functional cellular changes in in-vitro angiogenesis, and proliferation. Analysis of patient datasets demonstrates that SINHCAF and HIF 2 mRNA levels are inversely correlated and predict contrasting outcomes for patient survival in both colon and lung cancer. This relationship is also observed in a mouse model of colon cancer, indicating an evolutionary conserved mechanism. Our analysis reveals an unexpected link between SINHCAF and cancer cell signalling via regulation of the hypoxia response that is predictive of poor patient outcome.

molecular biology↗

bioRxiv2016

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.

The yeast Chd1 protein acts to position nucleosomes across genomes. Here we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state the DNA binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavourable alignment of C-terminal DNA binding region in solution contributes to an auto-inhibited state.

molecular biology↗