Cell cycle progression in mammals is modulated by two ubiquitin ligase complexes, CRL4 and SCF, which facilitate degradation of chromatin substrates involved in the regulation of DNA replication. One member of the CRL4 complex, the WD-40 containing protein RepID (DCAF14/PHIP), selectively binds and activates a group of replication origins. Here we show that RepID recruits the CRL4 complex to chromatin prior to DNA synthesis, thus playing a crucial architectural role in the proper licensing of chromosomes for replication. In the absence of RepID, cells rely on the alternative ubiquitin ligase, SKP2-containing SCF, to progress through the cell cycle. RepID depletion markedly increases cellular sensitivity to SKP2 inhibitors, which triggered massive genome re-replication. Both RepID and SKP2 interact with distinct, non-overlapping groups of replication origins, suggesting that selective interactions of replication origins with specific CRL components execute the DNA replication program and maintain genomic stability by preventing re-initiation of DNA replication.

Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of ‘dormant’ potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates replication fork elongation. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability.

Background: TIP60 is involved in transcriptional regulation acting as a coactivator or corepressor. Also, TIP60 acetylates histones to modulate chromatin remodeling. Results: TIP60 enhances transcriptional activity of RelA/p65, which is the active subunit of NF-B through their physical interaction. Conclusion: TIP60 is critical cofactor of RelA/p65 for transcription of NF-B target genes. Significance: TIP60 is involved in the NF-B pathway in hepatocarcinoma cells.

Cullin-RING (Really Interesting New Gene) E3 ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases, are functional multi-subunit complexes including substrate receptors, adaptors, cullin scaffolds, and RING-box proteins. CRLs are responsible for ubiquitination of ~20% of cellular proteins and are involved in diverse biological processes including cell cycle progression, genome stability, and oncogenesis. Not surprisingly, cullins are deregulated in many diseases and instances of cancer. Recent studies have highlighted the importance of CRL-mediated ubiquitination in the regulation of DNA replication/repair, including specific roles in chromatin assembly and disassembly of the replication machinery. The development of novel therapeutics targeting the CRLs that regulate the replication machinery and chromatin in cancer is now an attractive therapeutic strategy. In this review, we summarize the structure and assembly of CRLs and outline their cellular functions and their diverse roles in cancer, emphasizing the regulatory functions of nuclear CRLs in modulating the DNA replication machinery. Finally, we discuss the current strategies for targeting CRLs against cancer in the clinic.

The last decade has revealed new roles for Cullin-RING ubiquitin ligases (CRLs) in a myriad of cellular processes, including cell cycle progression. In addition to CRL1, also named SCF (SKP1-Cullin 1-F box protein), which has been known for decades as an important factor in the regulation of the cell cycle, it is now evident that all eight CRL family members are involved in the intricate cellular pathways driving cell cycle progression. In this review, we summarize the structure of CRLs and their functions in driving the cell cycle. We focus on how CRLs target key proteins for degradation or otherwise alter their functions to control the progression over the various cell cycle phases leading to cell division. We also summarize how CRLs and the anaphase-promoting complex/cyclosome (APC/C) ligase complex closely cooperate to govern efficient cell cycle progression.

Background: Next-generation sequencing allows genome-wide analysis of changes in chromatin states and gene expression. Data analysis of these increasingly used methods either requires multiple analysis steps, or extensive computational time. We sought to develop a tool for rapid quantification of sequencing peaks from diverse experimental sources and an efficient method to produce coverage tracks for accurate visualization that can be intuitively displayed and interpreted by experimentalists with minimal bioinformatics background. We demonstrate its strength and usability by integrating data from several types of sequencing approaches. Results: We have developed BAMscale, a one-step tool that processes a wide set of sequencing datasets. To demonstrate the usefulness of BAMscale, we analyzed multiple sequencing datasets from chromatin immunoprecipitation sequencing data (ChIP-seq), chromatin state change data (assay for transposase-accessible chromatin using sequencing: ATAC-seq, DNA double-strand break mapping sequencing: END-seq), DNA replication data (Okazaki fragments sequencing: OK-seq, nascent-strand sequencing: NS-seq, single-cell replication timing sequencing: scRepli-seq) and RNA-seq data. The outputs consist of raw and normalized peak scores (multiple normalizations) in text format and scaled bigWig coverage tracks that are directly accessible to data visualization programs. BAMScale also includes a visualization module facilitating direct, on-demand quantitative peak comparisons that can be used by experimentalists. Our tool can effectively analyze large sequencing datasets (~ 100 Gb size) in minutes, outperforming currently available tools. Conclusions: BAMscale accurately quantifies and normalizes identified peaks directly from BAM files, and creates coverage tracks for visualization in genome browsers. BAMScale can be implemented for a wide set of methods for calculating coverage tracks, including ChIP-seq and ATAC-seq, as well as methods that currently require specialized, separate tools for analyses, such as splice-aware RNA-seq, END-seq and OK-seq for which no dedicated software is available. BAMscale is freely available on github (https ://githu b.com/ncbi/BAMsc ale).

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.

Summary Caldesmon (CaD), which was originally identified as an actin-regulatory protein, is involved in the regulation of diverse actin-related signaling processes, including cell migration and proliferation, in various cells. The cellular function of CaD has been studied primarily in the smooth muscle system; nothing is known about its function in skeletal muscle differentiation. In this study, we found that the expression of CaD gradually increased as differentiation of C2C12 myoblasts progressed. Silencing of CaD inhibited cell spreading and migration, resulting in a decrease in myoblast differentiation. Promoter analysis of the caldesmon gene (Cald1) and gel mobility shift assays identified Sox4 as a major trans-acting factor for the regulation of Cald1 expression during myoblast differentiation. Silencing of Sox4 decreased not only CaD protein synthesis but also myoblast fusion in C2C12 cells and myofibril formation in mouse embryonic muscle. Overexpression of CaD in Sox4-silenced C2C12 cells rescued the differentiation process. These results clearly demonstrate that CaD, regulated by Sox4 transcriptional activity, contributes to skeletal muscle differentiation.