Acute depletion of human core nucleoporin reveals direct roles in transcription control but dispensability for 3D genome organization.

The nuclear pore complex (NPC) comprises more than 30 nucleoporins (NUPs) and is a hallmark of eukaryotes. NUPs have been suggested to be important in regulating gene transcription and 3D genome organization. However, evidence in support of their direct roles remains limited. Here, by Cut&Run, we find that core NUPs display broad but also cell-type-specific association with active promoters and enhancers in human cells. Auxin-mediated rapid depletion of two NUPs demonstrates that NUP93, but not NUP35, directly and specifically controls gene transcription. NUP93 directly activates genes with high levels of RNA polymerase II loading and transcriptional elongation by facilitating full BRD4 recruitment to their active enhancers. dCas9-based tethering confirms a direct and causal role of NUP93 in gene transcriptional activation. Unexpectedly, in situ Hi-C and H3K27ac or H3K4me1 HiChIP results upon acute NUP93 depletion show negligible changesS2211-1247(22)01437-1 of 3D genome organization ranging from A/B compartments and topologically associating domains (TADs) to enhancer-promoter contacts.

Seeing is believing: tools to study the role of Rho GTPases during cytokinesis.

Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spatiotemporal control of RhoA GTPase. Cytokinesis failure can lead to changes in cell fate or aneuploidy, which can be detrimental during development and/or can lead to cancer. However, our knowledge of the pathways that regulate RhoA during cytokinesis is limited, and the role of other Rho family GTPases is not clear. This is largely because the study of Rho GTPases presents unique challenges using traditional cell biological and biochemical methods, and they have pleiotropic functions making genetic studies difficult to interpret. The recent generation of optogenetic tools and biosensors that control and detect active Rho has overcome some of these challenges and is helping to elucidate the role of RhoA in cytokinesis. However, improvements are needed to reveal the role of other Rho GTPases in cytokinesis, and to identify the molecular mechanisms that control Rho activity. This review examines some of the outstanding questions in cytokinesis, and explores tools for the imaging and control of Rho GTPases.

Reprogrammed marrow adipocytes contribute to myeloma-induced bone disease.

Osteolytic lesions in multiple myeloma are caused by osteoclast-mediated bone resorption and reduced bone formation. A unique feature of myeloma is a failure of bone healing after successful treatment. We observed adipocytes on trabecular bone near the resorbed area in successfully treated patients. Normal marrow adipocytes, when cocultured with myeloma cells, were reprogrammed and produced adipokines that activate osteoclastogenesis and suppress osteoblastogenesis. These adipocytes have reduced expression of peroxisome proliferator-activated receptor γ (PPARγ) mediated by recruitment of polycomb repressive complex 2 (PRC2), which modifies PPARγ promoter methylation at trimethyl lysine-27 histone H3. We confirmed the importance of methylation in the PPARγ promoter by demonstrating that adipocyte-specific knockout of EZH2, a member of the PRC2, prevents adipocyte reprogramming and reverses bone changes in a mouse model. We validated the strong correlation between the frequency of bone lesions and the expression of EZH2 in marrow adipocytes from patients in remission. These results define a role for adipocytes in genesis of myeloma-associated bone disease and that reversal of adipocyte reprogramming has therapeutic implications.

Hotspot KRAS exon 2 mutations in CD166 positive colorectal cancer and colorectal adenoma cells.

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer deaths worldwide. Recent studies have shown that cancer stem cells (CSCs) are an important cause of tumor recurrence and metastasis. We hypothesized that CSCs marker CD166-positive CRC and colorectal adenoma (CAD) cells consist of more hotspot mutations than CD166-negative CRC and colorectal adenoma cells. To verify this, formalin fixed paraffin embedded tissue specimens from 42 patients each with CRC and CAD were recruited and CD166 immunohistochemical (IHC) staining followed by macrodissection was performed. DNA extracted was used for quantitative polymerase chain reaction detection on a somatic mutation array. Results showed that the immunoreactivity of CD166 protein had significant difference among CRC, CAD, and normal colorectal epithelial tissues (NCET) (P < 0.0001, Kruskal-Wallis test). Moreover, nucleotide changes were found in APC, KRAS, P53, PIK3CA, FBXW7 and SRC genes. Among those genes, KRAS exon 2 mutations were validated in another cohort of 70 CRC and 72 CAD specimens. Results showed that the difference in percentage of KRAS exon 2 mutations between CD166 positive and CD166 negative CRC specimens was significant (P < 0.05, chi-square test). Long term follow-up of the CRC patients showed that CD166-positive KRAS exon 2 mutations was useful in discriminating CRC patients with worse outcome. This study has provided evidence that KRAS exon 2 mutations are concentrated in CD166-positive cancer cells, with prognostic significance in CRC, and those mutations are also detected in CAD.

NanoString, a novel digital color-coded barcode technology: current and future applications in molecular diagnostics.

INTRODUCTION: Formalin-fixed, paraffin-embedded (FFPE) tissue sample is a gold mine of resources for molecular diagnosis and retrospective clinical studies. Although molecular technologies have expanded the range of mutations identified in FFPE samples, the applications of existing technologies are limited by the low nucleic acids yield and poor extraction quality. As a result, the routine clinical applications of molecular diagnosis using FFPE samples has been associated with many practical challenges. NanoString technologies utilize a novel digital color-coded barcode technology based on direct multiplexed measurement of gene expression and offer high levels of precision and sensitivity. Each color-coded barcode is attached to a single target-specific probe corresponding to a single gene which can be individually counted without amplification. Therefore, NanoString is especially useful for measuring gene expression in degraded clinical specimens. Areas covered: This article describes the applications of NanoString technologies in molecular diagnostics and challenges associated with its applications and the future development. Expert commentary: Although NanoString technology is still in the early stages of clinical use, it is expected that NanoString-based cancer expression panels would play more important roles in the future in classifying cancer patients and in predicting the response to therapy for better personal therapeutic care.

Genetic association between germline JAK2 polymorphisms and myeloproliferative neoplasms in Hong Kong Chinese population: a case-control study.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies that can be characterised by a somatic mutation (JAK2V617F). This mutation causes the bone marrow to produce excessive blood cells and is found in polycythaemia vera (~95%), essential thrombocythaemia and primary myelofibrosis (both ~50%). It is considered as a major genetic factor contributing to the development of these MPNs. No genetic association study of MPN in the Hong Kong population has so far been reported. Here, we investigated the relationship between germline JAK2 polymorphisms and MPNs in Hong Kong Chinese to find causal variants that contribute to MPN development. We analysed 19 tag single nucleotide polymorphisms (SNPs) within the JAK2 locus in 172 MPN patients and 470 healthy controls. Three of these 19 SNPs defined the reported JAK2 46/1 haplotype: rs10974944, rs12343867 and rs12340895. Allele and haplotype frequencies were compared between patients and controls by logistic regression adjusted for sex and age. Permutation test was used to correct for multiple comparisons. With significant findings from the 19 SNPs, we then examined 76 additional SNPs across the 148.7-kb region of JAK2 via imputation with the SNP data from the 1000 Genomes Project. RESULTS: In single-marker analysis, 15 SNPs showed association with JAK2V617F-positive MPNs (n = 128), and 8 of these were novel MPN-associated SNPs not previously reported. Exhaustive variable-sized sliding-window haplotype analysis identified 184 haplotypes showing significant differences (P < 0.05) in frequencies between patients and controls even after multiple-testing correction. However, single-marker alleles exhibited the strongest association with V617F-positive MPNs. In local Hong Kong Chinese, rs12342421 showed the strongest association signal: asymptotic P = 3.76 × 10-15, empirical P = 2.00 × 10-5 for 50,000 permutations, OR = 3.55 for the minor allele C, and 95% CI, 2.59-4.87. Conditional logistic regression also signified an independent effect of rs12342421 in significant haplotype windows, and this independent effect remained unchanged even with the imputation of additional 76 SNPs. No significant association was found between V617F-negative MPNs and JAK2 SNPs. CONCLUSION: With a large sample size, we reported the association between JAK2V617F-positive MPNs and 15 tag JAK2 SNPs and the association of rs12342421 being independent of the JAK2 46/1 haplotype in Hong Kong Chinese population.