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Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers — in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.

The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1, and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodeling module of the Nucleosome Remodeling and Deacetylase (NuRD) complex. To date, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential in defining underlying mechanisms of gene regulation. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS) we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodeling activity into both the ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner.We define a short aggregation prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the Nuclear Receptor Co-Repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

CCCTC-binding factor (CTCF) plays fundamental roles in transcriptional regulation and chromatin architecture maintenance. CTCF is also a tumour suppressor frequently mutated in cancer, however, the structural and functional impact of mutations have not been examined. We performed molecular and structural characterisation of five cancer-specific CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. Molecular docking and molecular dynamics confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding. However, R339Q and G420D were stabilised by the formation of new primary DNA bonds, contributing to gain-of-function. Our data confirm that a spectrum of loss-, change- and gain-of-function impacts on CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. Hence, diverse cellular phenotypes of mutant CTCF are clearly explained by examining structure–function relationships.

BackgroundHuman cancers commonly contain mutations in transcription factors that lead to aberrant DNA binding or altered effector function at target sites. One such factor significantly mutated in cancer is the evolutionarily-conserved CCCTC-binding factor (CTCF), which has fundamental roles in maintaining chromatin architecture and transcriptional regulation. Numerous cancer genome sequencing and functional studies have revealed CTCF’s role as a haploinsufficient tumour suppressor gene. However, to date, structure-function relationships of somatic CTCF mutations have not been examined.MethodsWe collated somatic CTCF mutations from cancer genome portals and published studies to determine their nature, frequency, distribution and potential functional impact. We undertook an in-depth examination of 5 CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. We performed functional analyses including cell growth, colony-formation, chromatin immunoprecipitation and transcriptional reporter assays. Based on their homology, each ZF mutation was then modelled on CTCF’s ZF domain crystal structure and its structural impact analysed using molecular dynamics simulations.ResultsWe observed an enrichment of somatic missense mutations occurring in the ZF region of CTCF, compared to the unstructured N- and C-termini. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. In silico mutagenesis revealed that L309P had the highest mutation energy and thus most severe impact on protein stability. Molecular docking and molecular dynamics simulations confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding (R339Q, R339W, R377H). Remarkably, R339Q and G420D were stabilised by the formation of new primary DNA bonds. All mutations exhibited some loss or gain of bonds at neighbouring residues, often in adjacent zinc fingers.ConclusionsOur data confirm the significant negative impact haploinsufficient CTCF ZF mutations have on its tumour suppressor function. A spectrum of loss-, change- and gain-of-function impacts in CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. We have established that diverse cellular phenotypes in CTCF are explained by examining structure-function relationships.

Normal protein-protein interactions (normPPIs) occur with high fidelity to regulate almost every physiological process. In cancer, this highly organised and precisely regulated network is disrupted, hijacked or reprogrammed resulting in oncogenic protein-protein interactions (oncoPPIs). OncoPPIs, which can result from genomic alterations, are a hallmark of many types of cancers. Recent technological advances in the field of mass spectrometry (MS)-based interactomics, structural biology and drug discovery have prompted scientists to identify and characterise oncoPPIs. Disruption of oncoPPI interfaces has become a major focus of drug discovery programs and has resulted in the use of PPI-specific drugs clinically. However, due to several technical hurdles, studies to build a reference oncoPPI map for various cancer types have not been undertaken. Therefore, there is an urgent need for experimental workflows to overcome the existing challenges in studying oncoPPIs in various cancers and to build comprehensive reference maps. Here, we discuss the important hurdles for characterising oncoPPIs and propose a three-phase multidisciplinary workflow to identify and characterise oncoPPIs. Systematic identification of cancertype-specific oncogenic interactions will spur new opportunities for PPI-focused drug discovery projects and precision medicine.

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