Gene expression and cell identity controlled by anaphase-promoting complex.

Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks1. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle2-5 but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters6,7, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division.

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding.

The yeast INO80 chromatin remodeling complex plays essential roles in regulating DNA damage repair, replication, and promoter architecture. INO80's role in these processes is likely related to its ability to slide nucleosomes, but the underlying mechanism is poorly understood. Here we use ensemble and single-molecule enzymology to study INO80-catalyzed nucleosome sliding. We find that the rate of nucleosome sliding by INO80 increases ∼100-fold when the flanking DNA length is increased from 40 to 60 bp. Furthermore, once sliding is initiated, INO80 moves the nucleosome rapidly at least 20 bp without pausing to re-assess flanking DNA length, and it can change the direction of nucleosome sliding without dissociation. Finally, we show that the Nhp10 module of INO80 plays an auto-inhibitory role, tuning INO80's switch-like response to flanking DNA. Our results indicate that INO80 is a highly processive remodeling motor that is tightly regulated by both substrate cues and non-catalytic subunits.

Principles of genome activation in the early embryo.

A major hurdle in an embryo's life is the initiation of its own transcriptional program, a process termed Zygotic Genome Activation (ZGA). In many species, ZGA is intricately timed, with bulk transcription initiating at the end of a series of reductive cell divisions when cell cycle duration increases. At the same time, major changes in genome architecture give rise to chromatin states that are permissive to RNA polymerase II activity. Yet, we still do not understand the series of events that trigger gene expression at the right time and in the correct sequence. Here we discuss new discoveries that deepen our understanding of how zygotic genes are primed for transcription, and how these events are regulated by the cell cycle and nuclear import. Finally, we speculate on the evolutionary basis of ZGA timing as an exciting future direction for the field.

Mitotic chromosomes scale to nuclear-cytoplasmic ratio and cell size in Xenopus.

During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.

Emergent properties of mitotic chromosomes.

As a cell prepares to divide, its genetic material changes dramatically in both form and function. During interphase, a dynamic interplay between DNA compartmentalization and transcription functions to program cell identity. During mitosis, this purpose is put on hold and instead chromosomes function to facilitate their accurate segregation to daughter cells. Chromatin loops are rearranged, stacked, and compressed to form X-shaped chromosomes that are neatly aligned at the center of the mitotic spindle and ready to withstand the forces of anaphase. Many factors that contribute to mitotic chromosome assembly have now been identified, but how the plethora of molecular mechanisms operate in concert to give rise to the distinct form and physical properties of mitotic chromosomes at the cellular scale remains under active investigation. In this review, we discuss recent work that addresses a major challenge for the field: How to connect the molecular-level activities to large-scale changes in whole-chromosome architecture that determine mitotic chromosome size, shape, and function.

Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones.

The hexameric AAA+ ATPases Rvb1 and Rvb2 (Rvbs) are essential for diverse processes ranging from metabolic signaling to chromatin remodeling, but their functions are unknown. While originally thought to act as helicases, recent proposals suggest that Rvbs act as protein assembly chaperones. However, experimental evidence for chaperone-like behavior is lacking. Here, we identify a potent protein activator of the Rvbs, a domain in the Ino80 ATPase subunit of the INO80 chromatin-remodeling complex, termed Ino80INS. Ino80INS stimulates Rvbs' ATPase activity by 16-fold while concomitantly promoting their dodecamerization. Using mass spectrometry, cryo-EM, and integrative modeling, we find that Ino80INS binds asymmetrically along the dodecamerization interface, resulting in a conformationally flexible dodecamer that collapses into hexamers upon ATP addition. Our results demonstrate the chaperone-like potential of Rvb1/Rvb2 and suggest a model where binding of multiple clients such as Ino80 stimulates ATP-driven cycling between hexamers and dodecamers, providing iterative opportunities for correct subunit assembly.

Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.