Mobility of kinetochore proteins measured by FRAP analysis in living cells

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

Orientation of mouse H19 ICR affects imprinted H19 gene expression through promoter methylation-dependent and -independent mechanisms

The mouse Igf2/H19 locus is regulated by genomic imprinting, in which the paternally methylated H19 imprinting control region (ICR) plays a critical role in mono-allelic expression of the genes in the locus. Although the maternal allele-specific insulator activity of the H19 ICR in regulating imprinted Igf2 expression has been well established, the detailed mechanism by which the H19 ICR controls mono-allelic H19 gene expression has not been fully elucidated. In this study, we evaluated the effect of H19 ICR orientation on imprinting regulation in mutant mice in which the H19 ICR sequence was inverted at the endogenous locus. When the inverted-ICR allele was paternally inherited, the methylation level of the H19 promoter was decreased and the H19 gene was derepressed, suggesting that methylation of the H19 promoter is essential for complete repression of H19 gene expression. Unexpectedly, when the inverted allele was maternally inherited, the expression level of the H19 gene was lower than that of the WT allele, even though the H19 promoter remained fully hypomethylated. These observations suggested that the polarity of the H19 ICR is involved in controlling imprinted H19 gene expression on each parental allele, dependent or independent on DNA methylation of the H19 promoter.

Gradual compaction of the central spindle decreases its dynamicity in PRC1 and EB1 gene-edited cells

During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent “bundling, sliding, and compaction” model of antiparallel midzone bundle formation in the central spindle during late mitosis.

Development and Comparative Evaluation of Endolysosomal Proximity Labeling-based Proteomic Methods in Human iPSC-derived Neurons

ABSTRACTProximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein-protein and protein-organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network, by knocking in (KI) an engineered ascorbate peroxidase (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batch of APEX labeling and different APEX probes. We conducted comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.

PRC1 and EB1 Binding Dynamics Reveal a Solidifying Central Spindle during Anaphase Compaction in Human Cells

ABSTRACTDuring mitosis the spindle undergoes considerable morphological and dynamic changes. Particularly the central spindle reorganizes drastically at the onset of anaphase when the antiparallel microtubule bundler PRC1 starts to accumulate and recruit a subset of spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using CRISPR/Cas9 gene editing, we generated human RPE1 cells that express from their endogenous locus fluorescently tagged versions of the two cytoskeletal network hub proteins PRC1 and the end binding protein EB1 to be able to quantify their spindle distribution and binding/unbinding turnover under native conditions. We find that throughout mitosis EB1 binds central spindle microtubule bundles in a PRC1-dependent manner using a binding mode different from EB1 at growing microtubule ends. Both proteins, PRC1 and EB1, progressively accumulate and bind increasingly strongly to compacting central antiparallel microtubule overlaps. These results show that the central spindle gradually ‘solidifies’ during mitosis, suggesting that the two protein interaction networks around PRC1 and EB1 cooperate to stabilize the shortening central spindle, explaining the importance of both proteins for correct chromosome segregation and cytokinesis.

Low Affinity Binding Sites in an Activating CRM Mediate Negative Autoregulation of the Drosophila Hox Gene Ultrabithorax

Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that maintains decreased Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in the proximal haltere in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.Author SummaryOne of the most fundamental questions in biology is how information encoded in the DNA is translated into the diversity of cell-types that exist within a multicellular organism, each with the same genome. Regulation at the transcriptional level, mediated through the activity of transcription factors bound to cis-regulatory modules (CRMs), plays a key role in this process. While we typically distinguish cell-type by the specific subset of genes that are transcriptionally ON or OFF, it is also important to consider the more fine-tuned transcriptional control of gene expression level. We focus on the regulatory logic of the Hox developmental regulator, Ultrabithorax (Ubx), in fruit flies, which exhibits both forms of transcriptional control. While ON/OFF control of Ubx is required to define differential appendage fate in the T2 and T3 thoracic segments, respectively, more fine-tuned control of transcription levels is observed in distinct compartments within the T3 appendage, itself, in which all cells exhibit a Ubx ON state. Through genetic analysis of regulatory inputs, and dissection of a Ubx CRM in a transgenic context and at the endogenous locus, we reveal a compartment-specific negative autoregulatory loop that dampens Ubx transcription to maintain distinct transcriptional levels within a single developing tissue.

Exploring the role of Polycomb recruitment in Xist-mediated silencing of the X chromosome in ES cells

Xist RNA has been established as the master regulator of X-chromosome inactivation (XCI) in female eutherian mammals but its mechanism of action remains unclear. By creating novel Xist mutants at the endogenous locus in mouse embryonic stem (ES) cells, we dissect the role of the conserved A-B-C-F repeats. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and 2 (PRC1/2) recruitment, which requires repeats B and C. Xist ΔB+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing.